Ffa1 (gpr40) as a therapeutic target for neural angiogenesis diseases or disorders

ABSTRACT

The instant invention provides methods and compositions related to discovery of Free Fatty Acid Receptor 1 (FFA1) as a therapeutic target for treatment or prevention of diseases or disorders of neurons that are characterized by angiogenesis, or of vascular diseases of the eye, retinal degeneration and/or tumors more generally. Therapeutic and/or prophylactic uses and compositions of known FFA1 inhibitors, including small molecules and nucleic acid agents, are described. Methods for identification of novel FFA1 inhibitors are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/999,535, filed Aug. 17, 2018, which is a national stage application,filed under 35 U.S.C. § 371, of International Application No.PCT/US2017/018418, filed Feb. 17, 2017, which claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.62/296,252, filed Feb. 17, 2016, the entire contents of all of which areincorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumbersEY024868, EY017017, EY022275, EY024963, and HD018655, awarded by theNational Institutes of Health. The Government has certain rights in theinvention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(C123370256US01-SEQ-RE.xml; Size: 32,043 bytes; and Date of Creation:Jan. 9, 2023) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Retinal neovascularization in the macula is the leading cause ofblindness in older adults (Lim, L. S., et al., Lancet 379, 1728-1738(2012)). Photoreceptors are amongst the highest energy consuming cellsof the body (Wong-Riley, M. T. T. Eye Brain 2, 99-116 (2010), and Okawa,H., et al., Curr Biol 18, 1917-1921 (2008)) and the macula is the regionof highest photoreceptor density.

Retinal angiomatous proliferation (RAP) vascular lesions are found inmacular telangiectasia (MacTel) (Yannuzzi, L. A., et al. Retina 32 Suppl1, 450460 (2012)) as well as in 15-20% of neovascular age-relatedmacular degeneration (AMD) (Bottoni, F., et al. Arch Ophthalmol 123,1644-1650 (2005)), consistent with high energy demands associated withretinal angiogenesis. Although VEGF contributes to neovascularization inmacular diseases, the factors that initiate VEGF secretion remainlargely unknown. Dyslipidimia and mitochondrial dysfunction (associatedwith aging) are important risk factors for neovascular AMD (Feehan, M.,et al. BMC Med Genet 12, 83 (2011), Fritsche, L. G., et al. Nat Genet40, 892-896 (2008), and Barot, M., et al., Curr Eye Res 36, 1069-1077(2011)). There is currently no cure for AMD or MacTel. Therefore, thereis a need in the field for the identification of therapeutics thatameliorate and/or prevent AMD and/or MacTel.

SUMMARY OF THE INVENTION

The invention is based, at least in part, upon the identification ofFree Fatty Acid Receptor 1 (FFA1, also known as G protein-coupledreceptor (GPR) 40-dependent (GPR40) protein) as a therapeutic target forneural cell (e.g., retinal cell) diseases and/or disorders that arecharacterized by angiogenesis. Targeting of FFA1 with one or moreantagonists, including known antagonists such as GW1100, antisenseand/or RNAi agents, for treatment or prevention of a disease or disorderof the eye (e.g., retina) characterized by angiogenesis is specificallycontemplated. In certain aspects of the invention, it is also identifiedthat targeting of FFA1 as described herein can exert a therapeuticeffect for vascular diseases of the eye such as age-related maculardegeneration (AMD) and retinopathy of prematurity (ROP), as well as forretinal degeneration and/or tumors in general. In related aspects, it isalso identified herein that not only FFA1 (GPR40), but also, e.g., GPR84and/or GPR 120 can be so targeted, with similar therapeutic effect.

Use of eye and/or retinal cells to screen for and identify additionalcompounds or agents that inhibit FFA1 is also contemplated. Withoutwishing to be bound by theory, inhibition of FFA1 is believed to exert atherapeutic effect by increasing glucose entry into the retina.

In one aspect, the invention provides a method for treating orpreventing angiogenesis in neural cells of a subject and/or treating orpreventing cancer in a subject, the method involving (a) identifying asubject having or at risk of neural cell angiogenesis and/or having orat risk of developing cancer; and (b) administering a FFA1 inhibitor tothe subject, thereby treating or preventing angiogenesis in the neuralcells of the subject and/or treating or preventing cancer in thesubject.

In one embodiment, the neural cells are retinal cells, optionallyphotoreceptor cells.

Another aspect of the invention provides a method for treating orpreventing retinal angiomatous proliferation (RAP) vascular lesions in asubject, the method involving (a) identifying a subject having or atrisk of developing RAP vascular lesions; and (b) administering a FFA1inhibitor to the subject, thereby treating or preventing RAP vascularlesions in the subject.

In certain embodiments, the subject has macular telangiectasia (MacTel)or neovascular age-related macular degeneration (AMD).

In one embodiment, the cells of the subject are impaired for lipiduptake, as compared to the cells of an appropriate control subject.

In another embodiment, the subject has dyslipidemia or mitochondrialdysfunction.

In an additional embodiment, the FFA1 inhibitor is a small moleculeantagonist or an RNAi agent.

Optionally, the FFA1 antagonist is GW1100.

In certain embodiments, the FFA1 inhibitor is administered to the eye ofthe subject.

Optionally, the FFA1 inhibitor is administered by intravitrealinjection.

In another embodiment, administering the FFA1 inhibitor enhances GLUT1expression in the retinal cells of the subject.

An additional aspect of the invention provides a method for increasingglucose uptake in a retinal cell, the method involving obtaining aretinal cell and contacting the retinal cell with a FFA1 inhibitor,thereby increasing glucose uptake in the retinal cell.

In one embodiment, the retinal cell is a retinal cell in vitro.

In certain embodiments, GLUT1 expression is enhanced in the retinal cellcontacted with the FFA1 inhibitor.

Another aspect of the invention provides a method for identifying a testcompound as a FFA1 inhibitor, the method involving contacting a retinalcell with a test compound; and measuring glucose uptake in the retinalcell, where measurement of increased glucose uptake in the retinal cellin the presence of the test compound identifies the test compound as aFFA1 inhibitor.

In certain embodiments, the retinal cell has a mutation or deletion ofthe very low-density lipoprotein receptor (Vldlr) gene that suppressesfatty acid uptake in the retinal cell.

In one embodiment, GLUT1 expression is enhanced in the retinal cellcontacted with the test compound.

An additional aspect of the invention provides a method for treating orpreventing a vascular disease of the eye, retinal degeneration and/orcancer in a subject, the method involving (a) identifying a subjecthaving or at risk of developing a vascular disease of the eye, retinaldegeneration and/or cancer; and (b) administering a GPR84 inhibitor tothe subject, thereby treating or preventing the vascular disease of theeye, retinal degeneration and/or cancer in the subject.

In one embodiment, the GPR84 inhibitor is a small molecule antagonist oran RNAi agent.

In another embodiment, the GPR84 inhibitor is GLPG1205.

In certain embodiments, the vascular disease of the eye is age-relatedmacular degeneration (AMD) or retinopathy of prematurity (ROP).

Another aspect of the invention provides a method for treating orpreventing a vascular disease of the eye, retinal degeneration and/orcancer in a subject, the method involving (a) identifying a subjecthaving or at risk of developing a vascular disease of the eye, retinaldegeneration and/or cancer; and (b) administering a GPR120 inhibitor tothe subject, thereby treating or preventing the vascular disease of theeye, retinal degeneration and/or cancer in the subject.

In certain embodiments, the GPR120 inhibitor is a small moleculeantagonist or an RNAi agent.

In another embodiment, the GPR120 inhibitor is4-Methyl-N-9H-xanthen-9-yl-benzenesulfonamide.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991). As used herein, the following terms have the meaningsascribed to them below, unless specified otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

An “agent” is meant any small compound, antibody, nucleic acid molecule,or peptide or fragment thereof. An “agent” includes a “therapeuticagent” as defined herein below.

As used herein, “Age-related macular degeneration,” or “AMD” refers toan eye condition which causes a deterioration or breakdown of themacula, a small spot near the center of the retina and the part of theeye needed for sharp central vision. More specifically, thephotoreceptor cells within the macula die off slowly, thus accountingfor the progressive loss of vision.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

An “agonist” as used herein is a molecule which enhances the biologicalfunction of a protein. The agonist may thereby bind to the targetprotein to elicit its functions. However, agonists which do not bind theprotein are also envisioned. The agonist may enhance the biologicalfunction of the protein directly or indirectly. Agonists which increaseexpression of certain genes are envisioned within the scope ofparticular embodiments of the invention. Suitable agonists will beevident, to those of skill in the art. For the present invention it isnot necessary that the agonist enhances the function of the targetprotein directly. Rather, agonists are also envisioned which stabilizeor enhance the function of one or more proteins upstream in a pathwaythat eventually leads to activation of targeted protein. Alternatively,the agonist may inhibit the function of a negative transcriptionalregulator of the target protein, wherein the transcriptional regulatoracts upstream in a pathway that eventually represses transcription ofthe target protein.

An “antagonist” may refer to a molecule that interferes with theactivity or binding of another molecule, for example, by competing forthe one or more binding sites of an agonist, but does not induce anactive response.

Cancer, as used herein, can include the following types of cancer,breast cancer, biliary tract cancer; bladder cancer; brain cancerincluding glioblastomas and medulloblastomas; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer;gastric cancer; hematological neoplasms including acute lymphocytic andmyelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma;hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma;AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; skin cancer including melanoma. Kaposi's sarcoma,basocellular cancer, and squamous cell cancer; testicular cancerincluding germinal tumors such as seminoma, non-seminoma (teratomas,choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancerincluding thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. Other cancers will beknown to one of ordinary skill in the art.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

By “dyslipidemia” is meant by an abnormal amount of lipids in the blood.Dyslipidemias were traditionally classified by patterns of elevation inlipids and lipoproteins. A more practical system categorizesdyslipidemias as primary or secondary and characterizes them byincreases in cholesterol only (pure or isolated hypercholesterolemia),increases in TGs only (pure or isolated hypertriglyceridemia), orincreases in both cholesterol and TGs (mixed or combinedhyperlipidemias).

By “effective amount” is meant the amount of an agent required toameliorate the symptoms of a disease relative to an untreated patient.The effective amount of active agent(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

“Free fatty acid receptor 1” or “Ffa1” is also referred to as GPR40, aclass A G-protein couple receptor, encoded by the Ffar1 gene(NM_005303.2 mRNA; NP_005294.1 protein). FFA1 is natively activated bymedium to long chain fatty acids.

By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA,shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof,that when administered to a mammalian cell results in a decrease (e.g.,by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a targetgene. Chitosan compositions are useful for the delivery ofpolynucleotides, such as inhibitory nucleic acid molecules, useful forthe treatment or prevention of pathogen infection and related disease.Typically, a nucleic acid inhibitor comprises at least a portion of atarget nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. For example, an inhibitory nucleic acid molecule comprises atleast a portion of any or all of the nucleic acids delineated herein.

The term “macula” refers to a small area within the retina. The maculais the part of the retina that is responsible for central vision,allowing things to be seen clearly. Although only a small part of theretina, the macula is more sensitive to detail than the rest of theretina. Many older people develop macular degeneration as part of thebody's natural aging process. Symptoms of macular degeneration includeblurriness, dark areas or distortion in central vision, or evenpermanent loss in central vision. It usually does not affect side orperipheral vision.

The term, “mitochondrial disease” refers to a chronic, genetic disorderthat occurs when the mitochondria of the cell fail to produce enoughenergy for cell or organ function. There are many forms of mitochondrialdisease including, mitochondrial myopathy, diabetes mellitus, Leber'shereditary optic neuropathy, Leigh syndrome, neuropathy, ataxia,retinitis pigmentosa and ptosis (NARP), myoclonic epilepsy and raggedred fibers (MERRF) and mitochondrial myopathy, encephalomyopathy, lacticacidosis, stroke-like syndromes (MELAS).

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

By “photoreceptor cells” refers to the hulk of neurons in the retina.The photoreceptor cells capture light energy units (photons) andregister the events as electrical signals of the central nervous system.The signals are then relayed to intermediary layers of neurons in theretina that process and organize the information before it istransmitted along the optic nerve fibers to the brain.

By “PPARα” refers to peroxisome proliferator-activated receptor alpha, anuclear protein encoded by the PPARA gene. PPARα is a transcriptionfactor and a regulator of lipid metabolism in the liver. PPARα isprimarily activated through ligand binding, comprising, for example,librate drugs used to treat hyperlipidemia, and a diverse set ofinsecticides, herbicides, plasticizers, and organic solvents, which arecollectively termed peroxisome proliferators.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reference” is meant a standard or control, e.g., a standard orcontrol condition.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “siRNA” is meant a double stranded RNA. Optimally, an siRNA is 18,19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhangat its 3′ end. These dsRNAs can be introduced to an individual cell orto a whole animal; for example, they may be introduced systemically viathe bloodstream. Such siRNAs are used to downregulate mRNA levels orpromoter activity.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNAduplex wherein a portion of the siRNA is part of a hairpin structure(shRNA). In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. In some embodiments, the loopis 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpinstructure can also contain 3′ or 5′ overhang portions. In some aspects,the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides inlength. In certain aspects, a nucleotide sequence in a vector serves asa template for the expression of a small hairpin RNA, comprising a senseregion, a loop region and an antisense region. Following expression, thesense and antisense regions form a duplex. It is this duplex, formingthe shRNA, which hybridizes to, for example, the Ffar1 mRNA and reducesexpression of FFA1, inducing neo-angiogenesis.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

A “therapeutically effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. An effectiveamount can be administered in one or more administrations.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms (e.g., AMD,MacTel or other angiogenesis-associated disease or disorder of the eye,or of tumors in general) associated therewith. It will be appreciatedthat, although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

The terms “tumor,” “solid tumor,” “primary tumor,” and “secondary tumor”refer to carcinomas, sarcomas, adenomas, and cancers of neuronal originand, in fact, to any type of cancer which does not originate from thehematopoietic cells and in particular concerns: carcinoma, sarcoma,adenoma, hepatocellular carcinoma, hepatocellular carcinoma,hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroidcarcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, synovioma, Ewing's tumor,leiomyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hematoma,bile duct carcinoma, melanoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, retinoblastoma, multiple myeloma, rectalcarcinoma, thyroid cancer, head and neck cancer, brain cancer, cancer ofthe peripheral nervous system, cancer of the central nervous system,neuroblastoma, cancer of the endometrium, as well as metastasis of allthe above.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent to thoseskilled in the art from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a schematic showing that retinal energy deficits areassociated with vascular lesions in Vldlr^(−/−). Photoreceptors havehigh metabolic rates, when adequate nutrients meet their energy demands,HIF is degraded and VEGF is not produced. Less substrate for glycolysisand fatty acid β-oxidation may decrease production of the Krebs cyclemetabolite α-ketoglutarate, a co-factor of propyl hydroxylase (PHD) thattags HIF1α for degradation. HIF1α stabilization can trigger VEGFexpression in photoreceptors, stimulating the development of pathologicneovascular lesions.

FIG. 1B depicts images showing pathologic vessels in Vldlr^(−/−) retinasoriginated from the deep vascular plexus (DVP) and breached the outerplexiform layer (P12), extending towards photoreceptor outer segments(os) at P16; Scale: 200 μm. n=5 retinas.

FIG. 1C depicts images and a bar graph of Vldlr^(−/−) pups raised indarkness (n=10 retinas) compared to normal 12 hours light/dark cycle(Ctl: control, n=28) to increase retinal energy demands, scale: 1 mm(left), 0.5 mm (others). White spots label vascular lesions. (P=0.0031).

FIG. 1D depicts images showing mitochondrial volume quantified by 3Dreconstruction of retinal scanning electron microscopy (SEM) images;mitochondria within photoreceptors (pseudo-colored); n=23photoreceptors. Scale: 5 μm. P<0.0001.

FIG. 1E depicts bar graphs showing that retinal ATP level wassignificantly lower in Vldlr^(−/−) retina (n=6) compared to littermatecontrol WT (n=4); two-tailed Student t-test, **P<0.01, ***P<0.001.P=0.0026. Results are presented in as mean±SEM.

FIG. 2A depicts a graph showing oxygen consumption rate (OCR) of wildtype (WT) retinas provided with long-chain fatty acid (FA) palmitate inthe presence or absence of FA oxidation inhibitor, etomoxir (40 μM);n=6-8 retinas FIG. 2B depicts a bar graph showing maximal OCR of WTretinas provided with long-chain fatty acid (FA) palmitate or control(Ctl: bovine serum albumin or BSA) in the presence or absence of FAoxidation inhibitor, etomoxir (40 μM); n=6-8 retinas.

FIG. 2C depicts a bar graph showing circulating plasma palimate levelsin WT and Vldlr^(−/−) mice. N=7 WT, 13 Vldlr^(−/−) mice plasma samples.(n=WT: 7, Vldlr^(−/−): 13 retinas P<0.0001).

FIG. 2D depicts a heat map showing a metabolite array of FA β-oxidationlevels measured by LC/MS/MS; n=3 animal retinas.

FIG. 2E depicts a bar graph showing total acylcarnitine and freecarnitine levels (P=0.0014) measured by LC/MS/MS; n=3 animal retinas.

FIG. 2F depicts bar graphs showing that Cpt1a mRNA of intact retinas(left: P=0.0052) laser capture microdissection (LCM) retinal layers byqRT-PCR. ONL: outer nuclear; INL: inner nuclear (photoreceptors) andGCL: ganglion cell layers; n=3 animal retinas.

FIG. 2G depicts images and a bar graph showing that ¹⁸F-FDG microPET/CTscan revealed decreased glucose uptake in Vldlr^(−/−) retinas, confirmedby retinal gamma radioactivity counts; Scale; 4 mm, n=22 WT, 12Vldlr^(−/−) retinas; P=0.0116.

FIG. 2H depicts a bar graph showing that Glut1 mRNA (expression inintact retinas left, n=9 WT; 12 Vldlr^(−/−) retinas; P=0.0119) andretinal layers (right) by LCM and qRT-PCR (n=3 retinas) (Two-tailedStudent t-test (FIGS. 2C-2F and FIGS. 2G-2I) and one-way ANOVA withTukey post-hoc analysis (FIGS. 2B, 2F, and 2H); *P<0.05, **P<0.01,***P<0.001.

FIG. 2I depicts a bar graph and blot showing that Glut1 proteinexpression of intact WT and Vldlr^(−/−) retina; n=6 retinas P=0.03;results are presented as mean±SEM two-tailed Student t-test (FIGS. 2C-2Fand FIGS. 2G-2I) and one-way ANOVA with Tukey post-hoc analysis (FIGS.2B, 2F, and 2H); *P<0.05, **P<0.01, ***P<0.001.

FIG. 3A depicts a schematic showing that FFA1 modulates retinal glucoseuptake and RAP. Decreased lipid uptake in Vldlr^(−/−) retina increasedextracellular mid/long chain FA, the agonist of lipid sensor FFA1, whichwas associated with reduced Glut1 expression.

FIG. 3B depicts a bar graph showing that expression of FA sensing GPCRin WT and Vldlr^(−/−) intact retinas.

FIG. 3C depicts a bar graph showing that Ffar distribution in retinallayers by LCM (qRT-PCR). ONL: outer nuclear layer, INL: inner nuclearlayer, GCL: ganglion cell layer; n=3 animal retinas; FFA1 agonistGW9508.

FIG. 3D depicts a bar graph showing that glucose uptake (³H-2-DG tracer)(n=Ctl: 5-8 ctl, GW: 9-16 GW-treated retinas).

FIG. 3E depicts a bar graph and a blot showing that FFA1 agonist GW9508agonist Glut1 protein expression (n=12 retinas; P<0.0001).

FIG. 3F depicts images and a bar graph showing that the number ofRAP-like pathologic vascular lesions at P16 in WT and in Vldlr^(−/−)mice.

FIG. 3G depicts images showing that Ffar1 deletion in Vldlr^(−/−) mice(Vldlr^(−/−)/Ffar1^(−/−)) reestablished glucose uptake (¹⁸F-FDG; n=4retinas; scale: 4 mm, GW: n=11 vehicle ctl: n=7, P=0.0002)).

FIG. 3H depicts bar graphs and a blot showing that Ffar1 deletion inVldlr^(−/−) mice (Vldlr^(−/−)/Ffar1^(−/−)) increased Glut1 proteinexpression to WT levels (n=WT: 10, others 9 retinas).

FIG. 3I depicts images and a bar graph showing that Ffar1 deletion inVldlr^(−/−) mice (Vldlr^(−/−)/Ffar1^(−/−)) reduced the number ofRAP-like pathologic vascular lesions of WT (no lesions) and Vldlr^(−/−)mice compared to littermate Vldlr^(−/−)/Ffar1^(+/+) mice (P16; n=10retinas; P=0.0153). Two-tailed Student t-test (FIGS. 3E, 3F, and 3I) andone-way ANOVA with Dunnett's (FIGS. 3B, 3C, 3G, and 3H) or Tukey's (FIG.3D) post-hoc comparison; *P≤0.05, **P<0.01, ***P<0.001. Results arepresented as mean±SEM.

FIG. 4A depicts a schematic showing that fuel deficient Vldlr^(−/−)retina generated less α-Ketoglutarate and more Vegf. Dual shortage ofglucose and FA uptake reduced acetyl-coA in Vldlr^(−/−) retina(LC/MS/MS; n=WT: 11, Vldlr^(−/−): 15 animal retinas).

FIG. 4B depicts a bar graph showing dual shortage of pyruvate and FAuptake reduced acetyl-coA in Vldlr^(−/−) retina (LC/MS/MS; n=WT: 15,Vldlr^(−/−): 12 animal retinas; P=0.0032).

FIG. 4C depicts a bar graph showing dual shortage of glucose and FAuptake reduced acetyl-coA in Vldlr^(−/−) retina (LC/MS/MS; n=WT: 11,Vldlr^(−/−): 15 animal retinas; P=0.0069); estimated by measuringacetylcarnitine.

FIG. 4D depicts a bar graph showing TCA (Krebs) cycle intermediate α-KGin Vldlr^(−/−) retina (LC/MS/MS; n=WT: 11, Vldlr^(−/−): 15 animalretinas; P=0.0016). Together with oxygen (O₂), α-KG is an essentialco-activator of propyl-hydroxylase dehydrogenase (PHD) that tags HIF1αfor degradation by proline hydroxylation (hydroxyproline).

FIG. 4E depicts a bar graph showing levels of hydroxyproline residues inWT and Vldlr^(−/−) retinas were measured by LC/MS/MS; n=WT: 15,Vldlr^(−/−): 12 animal retinas; P=0.0004).

FIG. 4F depicts a blot and a bar graph showing Hif1α stabilization ofWT, Vldlr^(−/−) and Vldlr^(−/−)/Ffar1^(−/−) retinal nuclear extractions.Fibrillarin (Fbl) was used as a nuclear loading control (n=3 allgroups).

FIG. 4G depicts images showing that Hif1α retinal expression inVldlr^(−/−) photoreceptor layer (ONL; P12 retinal flat mounts) Scale:100 μm; left: extended focus; middle and right panels: 3D confocal IHC,n=3.

FIG. 4H depicts a bar graph showing that Vegfa was also secreted (P16,ELISA, n=6 retinas; n=3 retinas).

FIG. 4I depicts images showing that Vegfa was also secreted and 3Dconfocal IHC, n=3 retinas; scale 100 μm; left extended focus; middle andright panels 3D confocal IHC.

FIG. 4J depicts an a bar graph showing that human subjects with AMD,either retinal angiomatous proliferation (RAP, n=3) or choroidalneovascularization (CNV, n=7) had higher VEGFA vitreous levels by ELISAcompared to control subjects without pathologic neovessels (macularhole; n=8). Two-tailed Student t-test (FIGS. 4C and 4D), Mann Whitneytest (FIGS. 4B and 4E), and one-way ANOVAs with post-hocDunett's (FIGS.4F and 4G) or Tukey's multiple comparison (FIG. 4H); *P≤0.05, **P<0.01,***P<0.001. Results are presented as mean±SEM.

FIG. 5A depicts a bar graph showing that photoreceptor-selective Vldlrdepletion generates RAP-like lesions. Retinal Vldlr expression in WT(n=4), heterozygous (het) Vldlr^(+/−) (n=5) and Vldlr^(−/−) mice (n=5retinas; qRT-PCR); ns: not significant.

FIG. 5B depicts a bar graph showing triglycerides of Vldlr^(+/−) (Het)mice used to locally knockdown Vldlr in photoreceptors. n=WT: 7Vldlr^(−/−): 8 plasmas.

FIG. 5C depicts a bar graph showing palmitate plasma levels ofVldlr^(+/−) (Het) mice used to locally knockdown Vldlr inphotoreceptors. n=WT: 7 Vldlr^(+/−): 8 plasmas.

FIG. 5D depicts an image showing that AAV2 viral vector containing aphotoreceptor-specific hRK promoter was cloned to include a fluorescenteGFP and different shRNA against Vldlr (Cahill, G. F. N Engl J Med 282,668-675 (1970), Wong-Riley, M. T. T. Eye Brain 2, 99-116 (2010), andNiu, Y.-G. & Evans, R. D. J Lipids 2011, 189876 (2011).

FIG. 5E depicts an image showing that timing of sub-retinal vectorinjection (P1) and retina collections (P12, 16, 26).

FIG. 5F depicts an image showing retinal distribution of viral vectors.

FIG. 5G depicts an image showing retinal distribution of viral vectorsin photoreceptors (ONL). OS: outer segment, IS: inner segment, ONL:outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer;n=5 retinas.

FIG. 5H depicts a bar graph showing retinal Vldlr suppression by 3different shRNA in Vldlr^(+/−) mice (qRT-PCR; n=shCtl: 8, shRNA-1: 12,shRNA-2: 4, shRNA-3: 8 retinas).

FIG. 5I depicts images showing the development of some RAP-like lesionswhen Vldlr was selectively depleted in Vldlr^(+/−) mouse photoreceptors.DVP: deep vascular plexus, RPE: retinal pigment epithelium. n=5 retinas.Two-tailed Student t-test (FIGS. 5B and 5C) and one-way ANOVA withDunnett's post-hoc comparison (FIGS. 5A and 5H); *P<0.05, **P<0.01,***P<0.001.

FIG. 6A depicts a schematic showing that fatty acids and glucose fuelthe mouse retina. FIG. 6A depicts a schematic overview of lipid andglucose metabolism converging to produce acetyl-coA, which fuels theKrebs (or TCA) cycle and the electron transfer chain (ETC) to produceATP; ns: not significant.

FIG. 6B depicts a graph showing oxygen consumption rates (OCR) of exvivo retinas incubated with palmitate in the presence (n=8) or absence(n=6 retinas) of Etomoxir (40 μM).

FIG. 6C depicts a bar graph showing maximal OCR of ex vivo retinasincubated with palmitate in the presence (n=8) or absence (n=6 retinas)of Etomoxir (40 μM); P=0.0027.

FIG. 6D depicts a graph showing OCR of ex vivo retinas incubated withglucose (12 mM) and treated or not with 2-deoxyglucose (2-DG; 100 mM) toinhibit glycolysis and glucose oxidation; n=8 retinas.

FIG. 6E depicts a bar graph showing maximal OCR of ex vivo retinasincubated with glucose (12 mM) and treated or not with 2-deoxyglucose(2-DG; 100 mM) to inhibit glycolysis and glucose oxidation. n=8 retinas;P=0.0019.

FIG. 6F depicts a bar graph showing maximal oxidation capacity forglucose (n=8) or palmitate (n=6 retinas) relative to their respectiveinhibitor.

FIG. 6G depicts a bar graph showing glucose uptake and lactate secretedby ex vivo retinas incubated in glucose-containing media (12 mM, 6hours); glycolysis accounted for the majority of 16 glucose utilization,producing 2 lactates per glucose molecule; n=4 retinas.

FIG. 6H depicts a bar graph showing that palmitate increased maximal OCRin wild type (WT; n=Ctl: 15, Palmitate: 14 retinas) but not inVldlr^(−/−) retinas (n=Ctl: 10, Palmitate: 13 retinas; P=0.0035);two-tailed Mann Whitney (FIGS. 6B, 6C, and 6F) or Student t-test (FIGS.6D, 6E, and 6H); **P<0.01.

FIG. 7A depicts an image and a bar graph showing deficient lipid uptakein Vldlr^(−/−) mice. Vldlr is highly expressed in photoreceptors (outernuclear layer; ONL) by laser capture microdissection (LCM and qRT-PCR).GCL: ganglion cell layer, INL: inner nuclear layer, RPE: retinal pigmentepithelium. n=3 retinas.

FIG. 7B depicts an image showing increased fluorochrome-labeledlong-chain FA (Bodipy C-16) in photoreceptor inner segments (IS) of WTmice, compared to Vldlr^(−/−) mice gavaged with these lipids. Also ofnote was visibly turbid serum in Vldlr^(−/−) mice from increased serumlipid associated with decreased lipid uptake (corner); n=3 retinas.

FIG. 7C depicts a bar graph showing reduced bromopalmitate-¹⁴C retinaluptake in Vldlr^(−/−) (n=12) compared to WT retinas (n=16).

FIG. 7D depicts a bar graph showing reduced bromopalmitate-¹⁴C retinaluptake in Vldlr^(−/−) (n=12) compared to WT retinas (n=16), associatedwith increased triglycerides.

FIG. 7E depicts an image and a bar graph showing reducedbromopalmitate-¹⁴C retinal uptake in Vldlr^(−/−) (n=12) compared to WTretinas (n=16), FA plasma levels (red: palmitate, C16); n=WT: 7,Vldlr^(−/−); 13 plasma samples; two-tailed Mann Whitney (FIG. 7E) orStudent t-test (FIGS. 7C-7E) and one-way ANOVA with Tukey's post-hoccomparison (FIG. 7A); *P<0.05, **P<0.01, ***P<0.001.

FIG. 8A depicts a bar graph showing the role of PPARα (peroxisomeproliferator-activated receptor a) in Vldlr^(−/−) mice. PPARα, whichregulates FA β-oxidation, was suppressed in Vldlr^(−/−) retina (n=3,P=0.0079).

FIG. 8B depicts a bar graph showing that PPARα, which regulates FAβ-oxidation, was suppressed in Vldlr^(−/−) retina mostly inphotoreceptors (ONL, n=3 animals).

FIG. 8C depicts images and a bar graph showing PPARα agonist WY16463reduced the number of vascular lesions. Ctl: n=10, WY: n=12 retinas;P=0.0429. Two-tailed Student t-test (FIGS. 8A and 8C) and one-way ANOVAwith Tukey's posthoc comparison (FIG. 8B); *P<0.05, **P<0.001.

FIG. 9A depicts a graph showing FA oxidation of exogenous and endogenouslipids by photoreceptors. In vitro oxygen consumption rates (OCR) ofphotoreceptors (661W) incubated with palmitate conjugated to BSA or BSAalone (control) in the presence or absence of Etomoxir (40 μM); n=5-6retinas.

FIG. 9B depicts a bar graph showing maximal oxidative capacity ofphotoreceptors (661W) incubated with palmitate conjugated to BSA or BSAalone (control) in the presence or absence of Etomoxir (40 IM); n=5-6retinas.

FIG. 9C depicts a graph showing OCR of photoreceptors (661 W) in thepresence or absence of palmitate and treated or not with PPARα agonist(GW9578, 100 nM, 48 hours); n=4-6 retinas.

FIG. 9D depicts a bar graph showing maximal oxidation capacity ofphotoreceptors (661W) in the presence or absence of palmitate andtreated or not with PPARα agonist (GW9578, 100 nM, 48 hours); n=4-6retinas.

FIG. 9E depicts a graph showing extra cellular acidification rate (ECAR)of photoreceptors (661W) in the presence or absence of palmitate andtreated or not with PPARα agonist (GW9578, 100 nM, 48 hours); n=4-6retinas.

FIG. 9F depicts a bar graph showing extra cellular acidification rate(ECAR) of photoreceptors (661W) in the presence or absence of palmitateand treated or not with PPARα agonist (GW9578, 100 nM, 48 hours); n=4-6retinas. PPARα agonist induced FA β-oxidation without significantlyaffecting glycolysis, as suggested by comparable acidification rates.One-way ANOVA with Tukey's post-hoc comparison (FIGS. 9A-9D) andKruskal-Wallis with Dunn's Multiple comparison (FIGS. 9E and 9F); ns:not significant, ***P<0.001.

FIG. 10A depicts an image showing that glucose metabolism was suppressedin Vldlr^(−/−) retina. Carbohydrate metabolism was the molecular pathwaymost regulated on a gene array comparing WT and Vldlr^(−/−) retinas.Ingenuity Pathway analysis, n=3 animals (littermates).

FIG. 10B depicts a bar graph showing pyruvate kinase (Pkm2), associatedwith the final unidirectional step of glycolysis, was highly regulatedon the gene array. Pkm2 suppression was confirmed in Vldlr^(−/−) retinaby qRT-PCR. n=WT: 6, Vldlr^(−/−): 5 retinas; P=0.0215.

FIG. 10C depicts a bar graph and a blot showing that Glut3 and 4 proteinexpression was not significantly different between WT (n=4) andVldlr^(−/−) (n=3) retinas; two-tailed Student t-test; *P<0.05.

FIG. 11A depicts a bar graph showing that Glut1 is regulated by FFA1.FFA1 agonist GW9508 (GW) reduces Glut1 expression in WT P=0.0142 andVldlr^(−/−) P=0.0284; retina; n=11-16 retinas.

FIG. 11B depicts a bar graph showing that Glucose uptake (¹⁸F-FDG);Ffar1 deletion in Vldlr^(−/−) mice reestablished retinal glucose uptakeand Glut1 expression n=b: 6-17, c: 3-9 retinas.

FIG. 11C depicts a bar graph showing Glut1 mRNA expression in WT,Vldlr^(−/−), Vldlr^(−/−)/Ffar1^(−/−), and Ffar1^(−/−) retinas. Ffar1deletion in Vldlr^(−/−) mice reestablished retinal glucose uptake andGlut1 expression n=b: 6-17, c: 3-9 retinas.

FIG. 11D depicts a bar graph showing knock-down (in vitro) of Ffar1(using siRNA; P=0.0025).

FIG. 11E depicts a bar graph showing that knock-down of Ffar1 usingsiRNA prevented Glut1 suppression by GW9508 in photoreceptor cells(661W); n=3 experiments.

FIG. 11F depicts a bar graph showing that inhibition of MEK/ERKsignaling (PD: PD98059, 20 μM; P=0.0056) prevented FFA1-mediated Glut1suppression in 661W photoreceptors, but not JNK signaling (SP: SP600125,50 μM, P=0.0121); n=3-7. Two-tailed Student t-test (FIGS. 11A, 11D, and11F) or Mann Whitney (FIG. 11F) and one-way ANOVA with Dunnett's (FIGS.11B and 11C) or Bonferroni's post-hoc comparison (FIG. 11E); *P≤0.05,**P<0.01, ***P<0.001. Results are presented as mean±SEM.

FIG. 12A depicts an image showing that FFA1 agonists increased retinalangiomatous proliferation. FFA1 is activated by fatty acids with C>6(Briscoe et al. J. Biol. Chem 278: 11303-11311). Mice fed MCT (middlechain triglycerides; n=10 retinas) with C8-10 more than doubled thenumber of vascular lesions compared to control (normal saline; n=15retinas; P=0.0011).

FIG. 12B depicts bar graphs showing that mice fed MCT suppressed Glut1expression in Vldlr^(−/−) retinas; n=6 retinas; P=0.0231.

FIG. 12C depicts an image and a bar graph showing that selective FFA1agonist TAK-875 significantly increased the number of RAP-like lesions;Ctl: n=9, TAK: n=8 retinas; P=0.0035; scale: 1 mm. Results are presentedas mean±SEM. Two-tailed Student t-test; *P<0.05, **P<0.01.

FIG. 13A depicts a blot and a bar graph showing that FFA1 stabilizesHifα and promotes Vegfa secretion in photoreceptors. In vivo, FFA1agonist GW9508 (GW) stabilized Hifα in WT retinas; n=6 experiments;P=0.0044.

FIG. 13B depicts a blot and a bar graph showing that FFA1 agonist GW9508(GW) stabilized Hifα in Vldlr^(−/−) retinas; n=6 experiments; P=0.0411.

FIG. 13C depicts a blot and a bar graph showing that decreased glucoseuptake in GW9508-treated or glucose starved photoreceptors (661W), wasassociated with stabilized Hifα; n=3 experiments; P=0.0006.

FIG. 13D depicts a bar graph showing that decreased glucose uptake inGW9508-treated or glucose starved photoreceptors (661W), was associatedwith increased Vegfa expression; n=3 experiments.

FIG. 13E depicts a bar graph showing that decreased glucose uptake inGW9508-treated or glucose starved photoreceptors (661W), was associatedwith secretion (ELISA); n=3 experiments; P=0.0013. Two-tailed Studentt-test (FIGS. 13A, 13C and 13E) or Mann Whitney test (FIG. 13B) andTwo-way ANOVA with Bonferroni post-hoc comparison (FIG. 13D); *P<0.05,**P<0.01, ***P<0.001. Results are presented as mean±SEM.

FIG. 14A depicts a bar graph showing that macrophages surround mature(but not early immature) RAP-like lesions. Markers of macrophages (CD68)were not increased in the initial phase of Vldlr^(−/−) RAP-like lesiondevelopment (P12), even in pups raised in darkness that have morevascular lesions; n=7-13 retinas.

FIG. 14B depicts a bar graph showing that inflammatory cytokines (TNFα)were not increased in the initial phase of Vldlr^(−/−) RAP-like lesiondevelopment (P12), even in pups raised in darkness that have morevascular lesions; n=7-13 retinas.

FIG. 14C depicts an image showing macrophages/microglial cells (Iba1,green).

FIG. 14D depicts an image showing that macrophages/microglial cellssurround mature vascular legions as confirmed by confocal cross-sectionsof these lesions; n=5 retinas.

FIG. 14E depicts an image showing that macrophages/microglial cells didnot surround nascent RAP-like vessels close to the deep vascular plexus(DVP), as confirmed by confocal cross-sections of these lesions. n=5retinas. ONL: outer nuclear layer. Scale bar: FIG. 14E, 20 μm.Kruskal-Wallis with Dunn's multiple comparison; *P<0.05, **P<0.01.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, at least in part, upon the discovery that theretina uses fatty acid (FA) β-oxidation for energy, and, in particular,that a lipid sensor, FFA1, curbs glucose uptake when FAs are available.Discovery of such a role for FFA1, at least in part, indicates that aneural cell (e.g., retinal cell) disease or disorder that ischaracterized by angiogenesis can be effectively treated or prevented ina subject and/or model system via administration of a therapeuticallyeffective/prophylactically effective amount of a FFA1 inhibitor to thesubject and/or model system. Optionally, treatment of vascular diseasesof the eye such as age-related macular degeneration (AMD) andretinopathy of prematurity (ROP), as well as treatment of retinaldegeneration and/or tumors in general is also contemplated forinhibitors of the invention. In related aspects, not only FFA1 (GPR40),but also, e.g., GPR84 and/or GPR 120 can be targeted, to similartherapeutic effect. Use of known FFA1 inhibitors, including smallmolecule compounds and inhibitory nucleic acids is expresslycontemplated. Methods for identifying new FFA1 inhibitors via, e.g., invitro monitoring of glucose uptake of retinal cells (optionallycomprising Vldlr deletion) contacted with, e.g., a compound library, isalso contemplated.

Additional aspects and embodiments of the invention are described below.

Mechanism of Action

Without wishing to be bound by theory, the instant invention is believedto function in the following manner. Tissues with high metabolic ratesoften use lipid as well as glucose for energy, conferring a survivaladvantage during feast and famine (Cahill, G. F. N Eng J Med 282,668-675 (1970)). Current dogma suggests that high-energy consumingphotoreceptors depend on glucose (Wong-Riley, M. T. T. Eye Brain 2,99-116 (2010)). Very low-density lipoprotein receptor (VLDLR), expressedin tissues with a high metabolic rate, facilitates the uptake oftriglyceride-derived FA (Niu, Y.-G. & Evans, R. D. J Lipids 2011, 189876(2011)). Vldlr is present in photoreceptors (Dorrell, M. I., et al. JClin Invest 119, 611-623 (2009)). In Vldlr^(−/−) retinas, FFA1, sensinghigh circulating lipid levels despite decreased FA uptake (Goudriaan, J.R., et al. Journal of lipid research 45, 1475-1481 (2004)), suppressesglucose transporter Glut1. This impaired glucose entry intophotoreceptors results in a dual lipid/glucose fuel shortage andreduction in the Krebs cycle intermediate α-ketoglutarate (KG). Low α-KGlevels promote hypoxia-induced factor-1α (Hif1α) stabilization andvascular endothelial growth factor (Vegfa) secretion by starvedVldlr^(−/−) photoreceptors, attracting neovessels to supply fuel. Theseaberrant vessels invading normally avascular photoreceptors inVldlr^(−/−) retinas are reminiscent of retinal angiomatous proliferation(RAP), a subset of neovascular age-related macular degeneration (AMD)(Bottoni, F., et al. Arch Ophthalmol 123, 1644-1650 (2005)), associatedwith high vitreous VEGF levels in humans. Dysregulated lipid and glucosephotoreceptor energy metabolism may therefore be a driving force inneovascular AMD and other retinal diseases.

Retinal neovascularization (RAP) is seen in macular telangiectasia(MacTel) (Yannuzzi, L. A., et al. Retina 32 Suppl 1, 450-460 (2012)) aswell as in 15.20% of macular neovascular age-related maculardegeneration (AMD) (Bottoni, F., et al. Arch Ophthalmol 123, 1644-1650(2005)), the leading cause of blindness in older adults (Lim, L. S.,Lancet 379, 1728-1738 (2012)). Photoreceptors, densest in the macula,are amongst the highest energy consuming and mitochondria-rich cells(Wong-Riley, Eye Brain 2, 99-116 (2010), and Okawa, Curr Biol 18,1917-1921 (2008)) consistent with high-energy demands causing macularneovascularization. VEGF contributes to retinal neovascularization, butfactors that initiate VEGF secretion in macular disease remain largelyunknown. Disordered photoreceptor mitochondrial energy metabolism washypothesized drive aberrant angiogenesis in the normally avascularphotoreceptors in an attempt to increase fuel supply, consistent withdyslipidemia and mitochondrial dysfunction (associated with aging) beingimportant risk factors of neovascular AMD (Lim, L. S., Lancet 379,1728-1738 (2012).

Retinal neurons are thought to rely on glucose for fuel (Wong-Riley, EyeBrain 2, 99-116 (2010), and Cohen, L. H. & Noell, W. K. J Neurochem 5,253-276 (1960)). Glucose is metabolized to pyruvate (by glycolysis) andeither converted to lactate in cytosol, or oxidized into acetyl-CoA inmitochondria before entering the Krebs cycle to produce ATP. Inphotoreceptors, the major glucose transporter is GLUT1 (Mantych, G. J.,Endocrinology 133, 600-607 (1993), and Gospe, S. M., J Cell Sci 123,3639-3644 (2010)). Clinically, GLUT1 deficiency causes infantileseizures and developmental delay (Klepper, J. Epilepsia 49 Suppl 8,46-49 (2008)), highlighting the importance of glucose metabolism in thebrain. However, GLUT1 deficient individuals have normal visionsuggesting alternative retinal energy substrates, perhaps through lipidβ-oxidation.

Lipid β-oxidation is common in heart and skeletal muscle with highmetabolic rates, where abundant VLDLR facilitates fatty acid (FA) uptake(Lopaschuk, G. D., et al., Physiol Rev 90, 207-258 (2010)). VLDLR bindschylomicrons and enables cleavage of long-chain FA from triglycerides(TG) by lipoprotein lipase (Goudriaan, J. R., et al. J Lipid Res 45,1475-1481 (2004))(Goudriaan, J. R., et al. Journal of lipid research 45,1475-1481 (2004)). VLDLR fosters transcytosis of active lipoproteinlipase across endothelial cells (Obunike, J. C., et al. J Biol Chem 276,8934-8941 (2001)), to deliver free FA to tissue, Vldlr deletionsuppresses lipid uptake and FA β-oxidation in the heart (Niu, Y.-G. &Evans, R. D. J Lipids 2011, 189876 (2011)). A similar mechanism inVldlr-rich and lipid-rich photoreceptors was hypothesized. Lipidβ-oxidation enzymes are expressed in the eye (Tyni, T., et al., PediatrRes 56, 744-750 (2004)). Clinically, VLDLR detection causes maculopathy(Sarac, O., et al., Ophthalmic Genet 33, 249-252 (2012)) and Vldlr^(−/−)mice develop RAP-like retinal vascular lesions (FIG. 1A) (Dorrell, M.I., et al. J Clin Invest 119, 611-623 (2009)). Vldlr^(−/−) mice allowexploration of the hypothesis that lipids fuel photoreceptors and fueldeficiency promotes neovessels.

Age-Related Macular Degeneration (AMD)

AMD is a common eye condition and a leading cause of vision loss amongpeople age 50 and older. It causes damage to the macula, a small spotnear the center of the retina and the part of the eye needed for sharp,central vision, which lets us see objects that are straight ahead. Insome people, AMD advances so slowly that vision loss does not occur fora long time. In others, the disease progresses faster and may lead to aloss of vision in one or both eyes. As AMD progresses, a blurred areanear the center of vision is a common symptom. Over time, the blurredarea may grow larger or you may develop blank spots in your centralvision. Objects also may not appear to be as bright as they used to be.

AMD by itself does not lead to complete blindness, with no ability tosee. However, the loss of central vision in AMD can interfere withsimple everyday activities, such as the ability to see faces, drive,read, write, or do close work, such as cooking or fixing things aroundthe house.

Dyslipidemia

Dyslipidemia is characterized by an abnormal amount of lipids in theblood. In developed countries, most dyslipidemias are hyperlipidemiaswhich are often due to diet and lifestyle. Dyslipidemias weretraditionally classified by patterns of elevation in lipids andlipoproteins. A more practical system categorizes dyslipidemias asprimary or secondary and characterizes them by increases in cholesterolonly (pure or isolated hypercholesterolemia), increases in TGs only(pure or isolated hypertriglyceridemia), or increases in bothcholesterol and TGs (mixed or combined hyperlipidemias).

Dyslipidemia usually causes no symptoms but can lead to symptomaticvascular disease, including coronary artery disease (CAD), stroke, andperipheral arterial disease. High levels of TGs (>1000 mg/dL [>11.3mmol/L]) can cause acute pancreatitis. High levels of LDL can causearcus corneae and tendinous xanthomas at the Achilles, elbow, and kneetendons and over metacarpophalangeal joints.

Mitrochondrial Disease (Age-Related)

Mitochondrial disease is a chronic, genetic disorder that occurs whenthe mitochondria of the cell fail to produce enough energy for cell ororgan function. The incidence is about 1:4000 individuals in the US.There are many forms of mitochondrial disease including, mitochondrialmyopathy, diabetes mellitus. Leber's hereditary optic neuropathy, Leighsyndrome, neuropathy, ataxia, retinitis pigmentosa and ptosis (NARP),myoclonic epilepsy and ragged red fibers (MERRF) and mitochondrialmyopathy, encephalomyopathy, lactic acidosis, stroke-like syndromes(MELAS).

Diseases of the mitochondria appear to cause the most damage to cells ofthe brain, heart, liver, skeletal muscles, kidney and the endocrine andrespiratory systems. Depending on which cells are affected, symptoms mayinclude loss of motor control, muscle weakness and pain,gastro-intestinal disorders and swallowing difficulties, poor growth,cardiac disease, liver disease, diabetes, respiratory complications,seizures, visual/hearing problems, lactic acidosis, developmental delaysand susceptibility to infection.

Macula

The macula is made up of millions of light-sensing cells that providesharp, central vision. It is the most sensitive part of the retina,which is located at the back of the eye. The retina turns light intoelectrical signals and then sends these electrical signals through theoptic nerve to the brain, where they are translated into the images wesee. When the macula is damaged, the center of your field of view mayappear blurry, distorted, or dark.

MacTel (Macular Telangiectasia)

Macular telangiectasia is a disease in which the macula is affected,causing a loss of central vision. The macula is a small area in theretina (the light-sensitive tissue lining the back of the eye) that isresponsible for central vision, allowing fine details to be seenclearly. Macular telangiectasia develops when there are problems withthe tiny blood vessels around the fovea, the center of the macula. Thereare two types of macular telangiectasia (Type 1 and Type 2), and eachaffects the blood vessels differently. Macular telangiectasia may occuras a result of a retinal vascular disease or a systemic disease such asdiabetes or hypertension, but in many cases, clinical findings reveal noknown cause.

One serious complication of macular telangiectasia is the development ofabnormal blood vessels under the retina. This is called chomidalneovascularization, and may call for injections of a drug calledvascular endothelial growth factor inhibitors (anti-VEGF). Anti-VEGFmedication targets a specific chemical in the eye that causes abnormalblood vessels to grow under the retina. That chemical is called vascularendothelial growth factor, or VEGF. Blocking VEGF with medicationinjections reduces the growth of abnormal blood vessels, slows theirleakage, helps to reduce swelling of the retina, and in some cases mayimprove vision.

Type 1 Macular Telangiectasia

In Type 1 macular telangiectasia, the blood vessels become dilatedforming tiny aneurysms, causing swelling and damaging macular cells. Thedisease almost always occurs in one eye, which differentiates it fromType 2.

Type 2 Macular Telangiectasia

The most common form of macular telangiectasia is Type 2 maculartelangiectasia, in which the tiny blood vessels around the fovea leak,become dilated (widen), or both. It is a bilateral disease of unknowncause, which characteristic alterations of macular capillary network andneurosensory atrophy. In some cases, new blood vessels form under theretina and they can also break or leak. Fluid from leaking blood vesselscauses the macula to swell or thicken, a condition called macular edema,which affects central vision. Also, scar tissue can sometimes form overthe macula and the fovea, causing loss of detail vision. Type 2 affectsboth eyes but not necessarily with the same severity.

RAP (Retinal Angiomatous Proliferation)

RAP describes a vascular process that originates within the neurosensoryretina, beginning with capillary proliferation, formation ofintraretinal neovascularization, and retinal-retinal anastamoses. RAPlesions have been characterized in the literature as having a poornatural history, but it is unclear what the reference group is. It isalso unclear whether these statements refer to vision outcomes, anatomicoutcomes, or both. Another recurring theme within the literaturereporting on RAP lesions is that these lesions do not respond well totreatment, and that no definite therapy has been shown to be beneficialat reducing visual loss and controlling the lesion. This has led to theuse of a variety of treatment modalities for these lesions, with thelist of therapies resembling those that have been used for anyAMD-related CNV lesion, including direct laser photocoagulation, transpupillary thermotherapy, surgical removal of the lesion, surgicalexcision of the retinal feeder vessels, photodynamic therapy (PDT)guided by fluorescein or indocyanine green (ICG) dye with and withoutintravitreal triamincolone, periocular anecortave acetate, anti-vascularendothelial growth factor (VEGF) regimens with pegaptanib sodium(EyeTech Pharmaceuticals, New York, USA), ranibizumab (Lucentis,Genentech Inc, South San Francisco, CA, USA), or bevacizumab (Avastin,Genentech Inc., South San Francisco, CA, USA), or various combinationsof the above.

Retinopathy of Prematurity (ROP)

Retinopathy of prematurity (ROP) is a potentially blinding eye disorderthat primarily affects premature infants weighing about 2¾ pounds (1250grams) or less that are born before 31 weeks of gestation (A full-termpregnancy has a gestation of 38-42 weeks). The smaller a baby is atbirth, the more likely that baby is to develop ROP. This disorder—whichusually develops in both eyes—is one of the most common causes of visualloss in childhood and can lead to lifelong vision impairment andblindness. ROP was first diagnosed in 1912.

With advances in neonatal care, smaller and more premature infants arebeing saved. These infants are at a much higher risk for ROP. Not allbabies who are premature develop ROP. There are approximately 3.9million infants born in the U.S. each year; of those, about 28,000 weigh2¾ pounds or less. About 14,000-16,000 of these infants are affected bysome degree of ROP. The disease improves and leaves no permanent damagein milder cases of ROP. About 90 percent of all infants with ROP are inthe milder category and do not need treatment. However, infants withmore severe disease can develop impaired vision or even blindness. About1,100-1,500 infants annually develop ROP that is severe enough torequire medical treatment. About 400-600 infants each year in the USbecome legally blind from ROP.

ROP is classified in five stages, ranging from mild (stage I) to severe(stage V):

-   -   Stage I—Mildly abnormal blood vessel growth. Many children who        develop stage I improve with no treatment and eventually develop        normal vision. The disease resolves on its own without further        progression.    -   Sta—ge II—Moderately abnormal blood vessel growth. Many children        who develop stage II improve with no treatment and eventually        develop normal vision. The disease resolves on its own without        further progression.    -   Stage III—Severely abnormal blood vessel growth. The abnormal        blood vessels grow toward the center of the eye instead of        following their normal growth pattern along the surface of the        retina. Some infants who develop stage III improve with no        treatment and eventually develop normal vision. However, when        infants have a certain degree of Stage III and “plus disease”        develops, treatment is considered. “Plus disease” means that the        blood vessels of the retina have become enlarged and twisted,        indicating a worsening of the disease. Treatment at this point        has a good chance of preventing retinal detachment.    -   Stage IV—Partially detached retina. Traction from the scar        produced by bleeding, abnormal vessels pulls the retina away        from the wall of the eye.    -   Stage V—Completely detached retina and the end stage of the        disease. If the eye is left alone at this stage, the baby can        have severe visual impairment anti even blindness. Most babies        who develop ROP have stages I or II. However, in a small number        of babies, ROP worsens, sometimes very rapidly. Untreated ROP        threatens to destroy vision.

Infants with ROP are considered to be at higher risk for developingcertain eye problems later in life, such as retinal detachment, myopia(nearsightedness), strabismus (crossed eyes), amblyopia (lazy eye), andglaucoma. In many cases, these eye problems can be treated orcontrolled.

ROP occurs when abnormal blood vessels grow and spread throughout theretina, the tissue that lines the back of the eye. These abnormal bloodvessels are fragile and can leak, scarring the retina and pulling it outof position. This causes a retinal detachment. Retinal detachment is themain cause of visual impairment and blindness in ROP.

Several complex factors may be responsible for the development of ROP.The eye starts to develop at about 16 weeks of pregnancy, when the bloodvessels of the retina begin to form at the optic nerve in the back ofthe eye. The blood vessels grow gradually toward the edges of thedeveloping retina, supplying oxygen and nutrients. During the last 12weeks of a pregnancy, the eye develops rapidly. When a baby is bornfull-term, the retinal blood vessel growth is mostly complete (Theretina usually finishes growing a few weeks to a month after birth). Butif a baby is born prematurely, before these blood vessels have reachedthe edges of the retina, normal vessel growth may stop. The edges of theretina—the periphery—may not get enough oxygen and nutrients.

It is believed that the periphery of the retina then sends out signalsto other areas of the retina fox nourishment. As a result, new abnormalvessels begin to grow. These new blood vessels are fragile and weak andcan bleed, leading to retinal scarring. When these scars shrink, theypull on the retina, causing it to detach from the back of the eye.

To date, the most effective proven treatments for ROP are laser therapyor cryotherapy. Laser therapy “burns away” the periphery of the retina,which has no normal blood vessels. With cryotherapy, physicians use aninstrument that generates freezing temperatures to briefly touch spotson the surface of the eye that overlie the periphery of the retina. Bothlaser treatment and cryotherapy destroy the peripheral areas of theretina, slowing or reversing the abnormal growth of blood vessels.Unfortunately, the treatments also destroy some side vision. This isdone to save the most important part of our sight—the sharp, centralvision we need for “straight ahead” activities such as reading, sewing,and driving.

Both laser treatments and cryotherapy are performed only on infants withadvanced ROP, particularly stage III with “plus disease.” Bothtreatments are considered invasive surgeries on the eye, and doctorsdon't know the long-term side effects of each.

In the inter stages of ROP, other treatment options include:

-   -   Scleral buckle. This involves placing a silicone band around the        eye and tightening it. This keeps the vitreous gel from pulling        on the scar tissue and allows the retina to flatten back down        onto the wall of the eye. Infants who have had a sclera buckle        need to have the band removed months or years later, since the        eye continues to grow; otherwise they will become nearsighted.        Sclera buckles are usually performed on infants with stage IV or        V.    -   Vitrectomy. Vitrectomy involves removing the vitreous and        replacing it with a saline solution. After the vitreous has been        removed, the scar tissue on the retina can be peeled back or cut        away, allowing the retina to relax and lay back down against the        eye wall. Vitrectomy is performed only at stage V.

While ROP treatment decreases the chances for vision loss, it does notalways prevent it. Not all babies respond to ROP treatment, and thedisease way get worse. If treatment for ROP does not work, a retinaldetachment may develop. Often, only part of the retina detaches (stageIV). When this happens, no further treatments may be needed, since apartial detachment may remain the same or go away without treatment.However, in some instances, physicians may recommend treatment to try toprevent further advancement of the retinal detachment (stage V). If thecenter of the retina or the entire retina detaches, central vision isthreatened, and surgery may be recommended to reattach the retina.

FFA1 (Also Referred to as GPR40)

By “Free fatty acid receptor 1” or “FFA1” is also referred to as GPR40,a class A G-protein couple receptor, encoded by the Ffar1 gene. FFA1 isactivated by medium to long chain fatty acids. GPCRs are membraneproteins characterized as having seven putative transmembrane domainsthat respond to a variety of molecules by activating intra-cellularsignaling pathways critical to a diversity of physiological functions.FFA1 was first identified as an orphan receptor (i.e., a receptorwithout a known ligand) from a human genomic DNA fragment. FFA1 ishighly expressed in pancreatic 1 cells and insulin-secreting cell lines.FFA1 activation is linked to modulation of the Gq family ofintra-cellular signaling proteins and concomitant induction of elevatedcalcium levels. It has been recognized that fatty acids serve as ligandsfor FFA1, and that fatty acids regulate insulin secretion through FFA1.Modulation (e.g., selective agonist stimulation) of the G-proteincoupled receptor results in phospholipase C activation, and productionof inositol 1,4,5-triphosphate and diacylglycerol, and increased levelsof intracellular calcium, which activates caspase-dependent apoptoticpathways (Tsujihata, et al., J Pharmacol Exp Ther 339: 228-237 (2011);and Briscoe, et al. J Bio Chem 278, 11303-11311 (2003)). GPR40 agonists(e.g., by selective agonist stimulation) have been used to inhibit thegrowth or induce apoptosis of certain cancer cells (e.g., cancersderived from neural crest tissues). Furthermore, GPR40 agonists havebeen reported to result in a cytotoxic effect on certain cancers andcancer cell lines. Agonist stimulation of GPR40 (e.g., via aomega-3fatty acids) activates signaling pathways that inhibit and are usefulfor the treatment and/or prevention of cancers.

GPCRs are membrane proteins having seven transmembrane domains, and canrespond to a variety of molecules, thereby activating intracellularsignaling transduction pathways, and are critical for achieving avariety of physiological functions. FFA1 was the first fatty acidreceptor to be identified on the cell surface, capable of binding themost common fatty acids in plasma such as palmitate, oleate, stearate,linoleate, and linolenate, and the like. FFA1 could be considered as anutrient sensing receptor, playing several tissue-dependent roles, whichmay affect overall glucose utilization and/or fat metabolism. Forexample, long-chain FFAs amplify GSIS in pancreatic 13 cells through theactivation of FFA1. A series of FFA1 agonists have been disclosed bysome patent applications, such as WO2005087710, WO2005051890, andWO2004106276.

Other GPCRs

Other GPCRs contemplated for targeting by the compositions, formulationsand methods of the instant invention include GPR 120, GPR44 (alsoreferred to as prostaglandin D₂ receptor 2 (DP₂), GPR42, GPR84, andhuman equivalents. GPR 120, for example, has been shown to mediate theanti-inflammatory and insulin-sensitizing effects of omega 3 fattyacids, and a deficiency of GPR120 is responsible for reduced fatmetabolism, thereby leading to obesity. An Exemplary GPR120 inhibitormay include 4-Methyl-N-9H-xanthen-9-yl-benzenesulfonamide.

In another example, GPR84 is a receptor for medium-chain FFA with carbonchain lengths of C9 to C14. Its expression is highly inducible ininflammation and its expression on neutrophils can be increased with LPSstimulation and reduced with GM-CSF stimulation. GPR84 is not activatedby short-chain and long-chain saturated and unsaturated FFAs induced inonocytes/macrophages by LPS. In addition, the activation of GPR84 inmonocytes/macrophages may amplify LPS stimulated IL-12 p40 production ina concentration dependent manner. An exemplary GPR 84 inhibitor isGLPG1205.

Diabetic Retinopathy

Diabetic retinopathy describes a diabetic eye disease that affects bloodvessels in the retina and is both the most common cause of vision lossamong people with diabetes and the leading cause of vision impairmentand blindness among working-age adults. Chronically high blood sugarfrom diabetes is associated with damage to the blood vessels in theretina, thereby leading to diabetic retinopathy. This changes thecurvature of the lens and results in the development of symptoms ofblurred vision. The blurring of distance vision as a result of lensswelling will subside once the blood sugar levels are brought undercontrol. Better control of blood sugar levels in patients with diabetesalso slows the onset and progression of diabetic retinopathy. Symptomsof diabetic retinopathy may include seeing spots or floaters in asubject's field of vision, blurred vision, having a dark or empty spotin the center of a subject's vision, and difficulty seeing well atnight. Diabetic retinopathy may progress through four stages:

-   -   I. Mild nonproliferative retinopathy: small areas of swelling in        the retinal blood vessels causing tiny bulges, called        microaneurysms to protrude from their walls may occur.    -   II. Moderate nonproliferative retinopathy: progression of the        disease may lead to blood vessels swelling and distorting,        therefore affecting their ability to transport blood.    -   III. Severe nonproliferative retinopathy: more blood vessels        become blocked, depriving the blood supply to areas of the        retina.    -   IV. Proliferative diabetic retinopathy: advanced stage of the        disease where growth factors secreted by the retina trigger the        proliferation of new blood vessels, which grow along the inside        surface of the retina and into the fluid that fills the eye. The        fragility of the new blood vessels makes them more likely to        leak and bleed. Scar tissue can cause retinal detachment        (pulling away of the retina from underlying tissue). Retinal        detachment can lead to permanent vision loss.

The fatty acid receptor GPR40 has drawn attention as a potentialtherapeutic target for treatment of type II diabetes. The art providessupport for the notion that activation of GPR40 improves glucosetolerance, which may in turn be beneficial for the treatment of type IIdiabetes (e.g., agonist TAK-875). However, the literature remainsunclear regarding whether inhibition of GPR40 would be beneficial totreat type II diabetes. A small molecule antagonist (DC260126), has beendemonstrated to protect against pancreatic β-cell dysfunction byreducing β-cell overload.

FFA1 Inhibitors

Inhibitors of FFA1 are contemplated for use in the methods andcompositions of the invention. Such inhibitors include both smallmolecules and nucleic acid inhibitory agents. Small molecule inhibitorsof FFA1 include the following.

GW-1100 (ethyl4-[5-[(2-ethoxypyrimidin-5-yl)methyl]-2-[(4-fluorophenyl)methylsulfanyl]-4-oxopyrimidin-1-yl]benzoate)is a known inhibitor of FFA1, having the following structure.

DC260126 (N-(4-Butylphenyl)-4-fluoro-benzenesulfonamide) is anothercompound identified as a small-molecule antagonist of GPR40 (Sun et al.PLOS DOI: 10.1371/journal.pone.0066744), possessing the followingstructure.

Other art-recognized inhibitors of FFA1, and of GPCRs more generallyinclude, e.g., pertussis toxin.

The above-described FFA1 inhibitory compounds are merely exemplary, asany art-recognized FFA1 inhibitor is contemplated for use in the methodsand compositions of the invention.

Neuronal Degeneration

Neuronal degeneration is characterized by a progressive loss ofstructure and function of neurons and including death of neurons. Manyneurodegenerative diseases including amyotrophic lateral sclerosis,Parkinson's Disease, Alzheimer's Disease, and Huntington's Disease occuras a result of neurodegenerative processes. Such diseases are incurable,resulting in progressive degeneration and/or death of neuron cells. Onlyan extremely small portion (less than 5%) of neurodegenerative diseasesare caused by genetic mutations and the remainder are caused by a buildup of toxic proteins in the brain and a loss of mitochondrial function,thereby leading to the increased levels of neurotoxic molecules. Asresearch progresses, many similarities appear that relate these diseasesto one another on a sub-cellular level. There are many parallels betweendifferent neurodegenerative disorders including atypical proteinassemblies as well as induced cell death. Neurodegeneration can be foundin many different levels of neuronal circuitry ranging from molecular tosystemic. The greatest risk factor for neurodegenerative diseases isaging. Mitochondrial DNA mutations as well as oxidative stress bothcontribute to aging. Many of these diseases are late-onset, and anunderlying factor in each disease is the gradual loss of function of theneurons with age. There are currently no therapies available to cureneurodegeneration. For each of the diseases, medication can onlyalleviate symptoms and help to improve patients' quality of life.

Cancer

The invention may further provide methods for the treatment of cancer,and more specifically may be used to alter the metabolism of malignantcells. Cancer may be referred to as an uncontrolled growth of cellswhich interferes with the normal functioning of the bodily systems.Cancers which migrate from their original location and seed vital organscan eventually lead to the death of the subject through the functionaldeterioration of the affected organs. Carcinomas are malignant cancersthat arise from epithelial cells and include adenocarcinoma and squamouscell carcinoma. Sarcomas are cancer of the connective or supportivetissue and include osteosarcoma, chondrosarcoma and gastrointestinalstromal tumor. Hematopoietic cancers, such as leukemia, are able tooutcompete the normal hematopoietic compartments in a subject, therebyleading to hematopoietic failure (in the form of anemia,thrombocytopenia and neutropenia) ultimately causing death. A person ofordinary skill in the art can classify a cancer as a sarcoma, carcinomaor hematopoietic cancer.

Cancer, as used herein, includes the following types of cancer, breastcancer, biliary tract cancer; bladder cancer; brain cancer includingglioblastomas and medulloblastomas; cervical cancer; choriocarcinoma;colon cancer; endometrial cancer; esophageal cancer; gastric cancer;hematological neoplasms including acute lymphocytic and myelogenousleukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cellleukemia; chromic myelogenous leukemia, multiple myeloma;AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; skin cancer including melanoma, Kaposi's sarcoma,basocellular cancer, and squamous cell cancer; testicular cancerincluding germinal tumors such as seminoma, non-seminoma (teratomas,choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancerincluding thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. Other cancers will beknown to one of ordinary skill in the art.

Inhibitory Nucleic Acids

A siRNA, shRNA or other inhibitory nucleic acid of the invention canalso be expressed from recombinant viral vectors intracellularly at ornear the area of neovascularization in vivo. The recombinant viralvectors of the invention comprise sequences encoding the siRNA, shRNA orother inhibitory nucleic acid of the invention and any suitable promoterfor expressing the siRNA, shRNA or other inhibitory nucleic acidsequences. Suitable promoters include, for example, the U6 or H1 RNA polIDI promoter sequences and the cytomegalovirus promoter. Selection ofother suitable promoters is within the skill in the art. The recombinantviral vectors of the invention can also comprise inducible orregulatable promoters for expression of the siRNA, shRNA or otherinhibitory nucleic acid in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver a siRNA, shRNA or other inhibitory nucleic acid of the inventionto cells in vivo is discussed in more detail below.

A siRNA, shRNA or other inhibitory nucleic acid of the invention can beexpressed from a recombinant viral vector either as two separate,complementary RNA molecules, or as a single RNA molecule with twocomplimentary regions.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operably linked.

In general, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of nucleic acid according to the invention. Viralvectors are an exemplary type of vector and include, but are not limitedto nucleic acid sequences from the following viruses: retrovirus, suchas moloney murine leukemia virus, harvey murine sarcoma virus, murinemammary tumor virus, and rous sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

Certain viral vectors are based on non-cytopathic eukaryotic viruses inwhich non-essential genes have been replaced with the gene of interest.Non-cytopathic viruses include retroviruses (e.g., lentivirus), the lifecycle of which involves reverse transcription of genomic viral RNA intoDNA with subsequent proviral integration into host cellular DNA.Retroviruses have been approved for human gene therapy trials. Mostuseful are those retroviruses that are replication-deficient (i.e.,capable of directing synthesis of the desired proteins, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell lined with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Murry, “Methods in Molecular Biology,” vol.7, Humana Press, Inc., Chiffon, N.J., 1991.

Certain viruses useful for delivery of nucleic acid agents of theinvention are the adenoviruses and adeno-associated (AAV) viruses, whichare double-stranded DNA viruses that have already been approved forhuman use in gene therapy. Actually 12 different AAV serotypes (AAV1 to12) are known, each with different tissue tropisms (Wu Z, Asokan A,Samulski R J: Adeno-associated virus serotypes: vector toolkit for humangene therapy. Mol Ther 14:316-327, 2006). Recombinant AAV are derivedfrom the dependent parvovirus AAV2 (Choi V W, Samulski R J, McCarty D M:Effects of adeno-associated virus DNA hairpin structure onrecombination. J Virol 79:6801-6807, 2005). The adeno-associated virustype 1 to 12 can be engineered to be replication deficient and iscapable of infecting a wide range of cell types and species (Wu Z,Asokan A, Samulski R J: Adeno-associated virus serotypes: vector toolkitfor human gene therapy. Mol Ther 14:316-327, 2006). It further hasadvantages such as, heat and lipid solvent stability; high transductionfrequencies in cells of diverse lineages, including hemopoietic cells;and lack of superinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression characteristic of retroviral infection. In addition,wild-type adeno-associated virus infections have been followed in tissueculture for greater than 100 passages in the absence of selectivepressure, implying that the adeno-associated virus genomic integrationis a relatively stable event. The adeno-associated virus can alsofunction in an extrachromosomal fashion and most recombinant adenovirusare extrachromosomal. In the sheltered environment of the retina, AAVvectors are able to maintain high levels of transgene expression in theretinal pigmented epithelium (RPE), photoreceptors, or ganglion cellsfor long periods of time after a single treatment. Each cell type can bespecifically targeted by choosing the appropriate combination of AAVserotype, promoter, and intraocular injection site (Dinculescu et al.,Hum Gene Ther. 2005 June; 16(6):649-63 and Lebherz, C., Maguire, A.,Tang, W., Bennett, J. & Wilson, J. M. Novel AAV serotypes for improvedocular gene transfer. J Gene Med 10, 375-82 (2008)). In one embodiment,AAV serotype 8 is particularly suitable.

Any viral vector capable of accepting the coding sequences for thesiRNA, shRNA or other inhibitory nucleic acid molecule(s) to beexpressed can be used, for example vectors derived from adenovirus (AV);adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV),Rhabdoviruses, murine leukemia virus); herpes virus, and the like. Thetropism of the viral vectors can also be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses. For example, an AAV vector of the invention can be pseudotypedwith surface proteins from vesicular stomatitis virus (VSV), rabies,Ebola, Mokola, and the like.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1998),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;and Anderson W F (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

Optionally, viral vectors derived from AV and AAV are employed. Incertain embodiments, the siRNA, shRNA or other inhibitory nucleic acidof the invention is expressed as two separate, complementarysingle-stranded RNA molecules from a recombinant AAV vector comprising,for example, either the U6 or H1 RNA promoters, or the cytomegalovirus(CMV) promoter.

A suitable AV vector for expressing the siRNA, shRNA or other inhibitorynucleic acid of the invention, a method for constructing the recombinantAV vector, and a method for delivering the vector into target cells, aredescribed in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the siRNA, shRNA or other inhibitorynucleic acid of the invention, methods for constructing the recombinantAAV vector, and methods for delivering the vectors into target cells aredescribed in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher KJ et al. (1996), J. Virol., 70: 520-532; Samulski R et al. (1989), J.Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; InternationalPatent Application No. WO 94/13788; and International Patent ApplicationNo. WO 93/24641, the entire disclosure of which are herein incorporatedby reference.

Non-viral administration of nucleic acid in vivo has been accomplishedby a variety of methods. These include lipofectin/liposome fusion ProcNatl Acad Sci 84, pp 7413-7417 (1993), polylysine condensation with andwithout adenovirus enhancement Human Gene Therapy 3, pp 147-154 (1992),and transferrin transferring receptor delivery of nucleic acid to cellsProc Natl Acad Sci 87, pp 3410-3414 (1990) The use of a specificcomposition consisting of polyacrylic acid has been disclosed in WO94/24983 Naked DNA has been administered as disclosed in WO90/11092.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of a nucleic acid of the inventioninto target cells.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Synthetic cationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a nucleic acid. The use ofcationic lipids may promote encapsulation of negatively charged nucleicacids, and also promote fusion with negatively charged cell membranes(Feigner et al., 1989).

Alternatively, one of the simplest and the safest ways to deliver thenucleic acid according across cell membranes in vivo may involve thedirect application of high concentration free or naked polynucleotides(typically mRNA or DNA). By “naked DNA (or RNA)” is meant a DNA (RNA)molecule which has not been previously complexed with other chemicalmoieties. Naked DNA uptake by animal cells may be increased byadministering the cells simultaneously with excipients and the nucleicacid. Such excipients are reagents that enhance or increase penetrationof the DNA across cellular membranes and thus delivery to the cellsdelivery of the therapeutic agent. Various excipients have beendescribed in the art, such as surfactants, e.g. a surfactant selectedform the group consisting of Triton X-100, sodium dodecyl sulfate, Tween20, and Tween 80; bacterial toxins, for instance streptolysin O, choleratoxin, and recombinant modified labile toxin of E coli; andpolysaccharides, such as glucose, sucrose, fructose, or maltose, forinstance, which act by disrupting the osmotic pressure in the vicinityof the cell membrane. Other methods have been described to enhancedelivery of free polynucleotides, such as blocking of polynucleotideinactivation via endo—or exonucleolytic cleavage by both extra—andintracellular nucleases.

In certain embodiments, a nucleic acid of the invention is under thecontrol of a heterologous regulatory region, e.g., a heterologouspromoter. The promoter can be a generally active promoter, or in certainembodiments, can be, e.g., an eye and/or photoreceptor specificpromoter, such as the three versions of the human red cone opsinpromoter (PRO.5, 3LCR-PRO.5 and PR2.1), the human blue cone opsinpromoter HB569 (Gene Ther. 2008 July; 15(14):1049-55. Epub 2008 Mar. 13,Targeting gene expression to cones with human cone opsin promoters inrecombinant AAV. Komáromy A M, Alexander J J, Cooper A E, Chiodo V A,Glushakova L G, Acland G M, Hauswirth W W, Aguirre G D); threephotoreceptor specific promoters (interphotoreceptor retinoid bindingprotein-IRPB1783; guanylate cyclase activating protein 1-GCAP292;rhodopsin-mOP500) ‘(Mol Vis. 2007 Oct. 18; 13:2001-11. Targetedexpression of two proteins in neural retina using self-inactivating,insulated lentiviral vectors carrying two internal independentpromoters. Semple-Rowland S L, Eccles K S, Humberstone E J.) the humanrhodopsin kinase (RK) promoter (Invest Ophthalmol Vis Sci. 2007September; 48(9):3954-61. AAV-mediated expression targeting of rod andcone photoreceptors with a human rhodopsin kinase promoter. Khani S C,Pawlyk B S, Bulgakov O V, Kasperek E, Young J E, Adamian M, Sun X, SmithA J, Ali R R, Li T.); the promoter for the alpha subunit of conetransducin or the cone photoreceptor regulatory element 1 (CPRE-1) anovel 20-bp enhancer element in the TalphaC promoter (J Biol Chem. 2008Apr. 18; 283(16):10881-91. Epub 2008 Feb. 13. A novel, evolutionarilyconserved enhancer of cone photoreceptor-specific expression. Smyth V A,Di Lorenzo D, Kennedy B N.), the promoter of the orphan nuclear receptorNr2e3; the promoter of human retinal guanylate cyclase 1 (retGC1), andthe cone transcription factor Trβ2 [(Peng and Chen, 2005; Oh et al.,2007) promoter for the beta subunit of the phosphodiesterase, PDE6B(Mali et al., 2007). The promoter can also optionally be selected formthe group of genes consisting of human rhodopsin (hRHO), human red opsin(hRO), human green opsin and mouse cone arrestin-3 (mCAR). In a certainembodiments, mouse cone arrestin-3 (mCAR) can be used.

Suitable methods, i.e., invasive and noninvasive methods, ofadministering a nucleic acid of the invention, optionally so as tocontact a photoreceptor, are well known in the art. Although more thanone route can be used to administer a nucleic acid, certain routes canprovide a more immediate and more effective reaction than other routes.Accordingly, the described routes of administration are merely exemplaryand are in no way limiting. Accordingly, the methods are not dependenton the mode of administering the nucleic acid of the invention to ananimal, optionally a human, to achieve the desired effect. As such, anyroute of administration is appropriate so long as the nucleic acid ofthe invention targets an appropriate host cell (e.g., an eye cell, e.g.,a retinal and/or retinal-associated cell involved in treating orpreventing angiogenesis upon FFA1 inhibition). A nucleic acid of theinvention can be appropriately formulated and administered in the formof an injection, eye lotion, ointment, implant and the like. A nucleicacid of the invention can be applied, for example, systemically,topically, subconjunctivally, intraocularly, retrobulbarly,periocularly, subretinally, or suprachoroidally. In certain cases, itmay be appropriate to administer multiple applications and employmultiple routes, e.g., subretinal and intravitreous, to ensuresufficient exposure of targeted eye cells (e.g., retinal cells and/orretina-associated cells) to the nucleic acid of the invention. Multipleapplications of the nucleic acid of the invention may also be requiredto achieve a desired effect.

Depending on the particular case, it may be desirable to non-invasivelyadminister a nucleic acid of the invention to a patient. For instance,if multiple surgeries have been performed, the patient displays lowtolerance to anesthetic, or if other ocular-related disorders exist,topical administration of the nucleic acid according to the inventionmay be most appropriate. Topical formulations are well known to those ofskill in the art. Such formulations are, suitable in the context of thepresent invention for application to the eye. The use of patches,corneal shields (see, e.g., U.S. Pat. No. 5,185,152), and ophthalmicsolutions (see, e.g., U.S. Pat. No. 5,710,182) and ointments, e.g., eyedrops, is also within the skill in the art. A nucleic acid of theinvention can also be administered non-invasively using a needlelessinjection device, such as the Biojector 2000 Needle-Free InjectionManagement System@ available from Bioject, Inc.

A nucleic acid of the invention can optionally be present in or on adevice that allows controlled or sustained release of the nucleic acidaccording, such as an ocular sponge, meshwork, mechanical reservoir, ormechanical implant. Implants (see, e.g., U.S. Pat. Nos. 5,443,505,4,853,224 and 4,997,652), devices (see, e.g., U.S. Pat. Nos. 5,554,187,4,863,457, 5,098,443 and 5,725,493), such as an implantable device,e.g., a mechanical reservoir, an intraocular device or an extraoculardevice with an intraocular conduit, or an implant or a device comprisedof a polymeric composition are particularly useful for ocularadministration of the nucleic acid according to the invention. A nucleicacid according to the invention can also be administered in the form ofsustained-release formulations (see, e.g., U.S. Pat. No. 5,378,475)comprising, for example, gelatin, chondroitin sulfate, apolyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), or apolylacticglycolic acid.

Alternatively, a nucleic acid according to the invention can beadministered using invasive procedures, such as, for instance,intravitreal injection or subretinal injection optionally preceded by avitrectomy. Subretinal injections can be administered to differentcompartments of the eye, i.e., the anterior chamber. While intraocularinjection is preferred, injectable compositions can also be administeredintramuscularly, intravenously, and intraperitoneally. Pharmaceuticallyacceptable carriers for injectable compositions are well-known to thoseof ordinary skill in the art (see Pharmaceutics and Pharmacy Practice,J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds.,pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel,41h ed., pages 622.630 (1986)). A nucleic acid according to theinvention can also be administered in vivo by particle bombardment,i.e., a gene gun. Optionally, a nucleic acid of the invention isadministered via an ophthalmologic instrument for delivery to a specificregion of an eye. Use of a specialized ophthalmologic instrument canensure precise administration of the nucleic acid while minimizingdamage to adjacent ocular tissue. Delivery of a nucleic acid of theinvention to a specific region of the eye also limits exposure ofunaffected cells to nucleic acid of the invention, thereby reducing therisk of side effects. An exemplary ophthalmologic instrument is acombination of forceps and subretinal needle or sharp bent cannula.Alternatively, a nucleic acid of the invention may be injected directlyinto the vitreous, aqueous humour, ciliary body tissue(s) or cellsand/or extra-ocular muscles by electroporation or iontophoresis means.

The dose of a nucleic acid of the invention administered to an animal,particularly a human, in accordance with the present invention should besufficient to effect the desired response in the animal over areasonable time frame. One skilled in the art will recognize that dosagewill depend upon a variety of factors, including the age, species, thepathology in question, and condition or disease state. Dosage alsodepends on the nucleic acid to be expressed and/or active, as well asthe amount of ocular tissue about to be affected or actually affected bythe neural cell (e.g., retinal cell) disease or disorder. The size ofthe dose also will be determined by the route, timing, and frequency ofadministration as well as the existence, nature, and extent of anyadverse side effects that might accompany the administration of aparticular nucleic acid according to the invention and the desiredphysiological effect. It will be appreciated by one of ordinary skilledin the art that various conditions or disease states, in particular,chronic conditions or disease states, may require prolonged treatmentinvolving multiple administrations.

A nucleic acid of the invention can be administered in a pharmaceuticalcomposition, which comprises a pharmaceutically acceptable carrier andthe nucleic acid(s) of the invention. Any suitable pharmaceuticallyacceptable carrier can be used within the context of the presentinvention, and such carriers are well known in the art. The choice ofcarrier will be determined, in part, by the particular site to which thecomposition is to be administered and the particular method used toadminister the composition.

Suitable formulations include aqueous and non-aqueous solutions,isotonic sterile solutions, which can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood or intraocular fluid of the intended recipient, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Theformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, immediately prior to use.Extemporaneous solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.Optionally, the pharmaceutically acceptable carrier is a buffered salinesolution. In certain embodiments, a nucleic acid of the invention foruse in the present inventive methods is administered in a pharmaceuticalcomposition formulated to protect the nucleic acid of the invention fromdamage prior to administration. For example, the pharmaceuticalcomposition can be formulated to reduce loss of the nucleic acid of theinvention on devices used to prepare, store, or administer the nucleicacid of the invention, such as glassware, syringes, or needles. Thepharmaceutical composition can be formulated to decrease the lightsensitivity and/or temperature sensitivity of the nucleic acid of theinvention. To this end, the pharmaceutical composition optionallycomprises a pharmaceutically acceptable liquid carrier, such as, forexample, those described above, and a stabilizing agent selected fromthe group consisting of polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such apharmaceutical composition will extend the shelf life of the nucleicacid, facilitate administration, and increase the efficiency of themethods of the invention. In this regard, a pharmaceutical compositionalso can be formulated to enhance transduction efficiency.

In addition, one of ordinary skill in the art will appreciate that anucleic acid can be present in a composition with other therapeutic orbiologically-active agents. For example, therapeutic factors useful inthe treatment of a particular indication can be present. For instance,if treating vision loss, hyaluronidase can be added to a composition toeffect the breakdown of blood and blood proteins in the vitreous of theeye. Factors that control inflammation, such as ibuprofen or steroids,can be part of the composition to reduce swelling and inflammationassociated with in vivo administration of the nucleic acid according tothe invention and ocular distress. Immune system suppressors can beadministered in combination to reduce any immune response to the nucleicacid itself. Similarly, vitamins and minerals, anti-oxidants, andmicronutrients can be co-administered. Antibiotics, i.e., microbicidesand fungicides, can be present to reduce the risk of infectionassociated with gene transfer procedures and other disorders.

The present invention also relates to pharmaceutical compositionscomprising an isolated nucleic acid according to the invention.

The present invention also relates to a method for treating a neuralcell (e.g., retinal cell) disease or disorder comprising administering apatient in need thereof with a therapeutically effective amount of anisolated nucleic acid according to the invention.

The ability of a siRNA, shRNA or other inhibitory nucleic acidcontaining a given target sequence to cause RNAi-mediated degradation ofthe target mRNA can be evaluated using standard techniques for measuringthe levels of RNA or protein in cells. For example, siRNA, shRNA orother inhibitory nucleic acid of the invention can be delivered tocultured cells, and the levels of target mRNA can be measured byNorthern blot or dot blotting techniques, or by quantitative RT-PCR.Alternatively, the levels of FFA1 protein in, e.g., cultured cellsand/or cells of a subject, can be measured by ELISA or Western blot.

RNAi-mediated degradation of target mRNA by a siRNA, shRNA or otherinhibitory nucleic acid containing a given target sequence can also beevaluated with animal models of retinal angiogenesis, such as the mousemodels described herein. For example, areas ofangiogenesis/neovascularization in a mouse can be measured before andafter administration of a siRNA, shRNA or other inhibitory nucleic acidof the invention. A reduction in the areas ofangiogenesis/neovascularization in such mice upon administration of thesiRNA, shRNA or other inhibitory nucleic acid indicates thedown-regulation of the target mRNA (e.g., Ffar1).

Pharmaceutical Compositions

Another aspect of the invention pertains to pharmaceutical compositionsof the compounds of the invention. The pharmaceutical compositions ofthe invention typically comprise a compound of the invention and apharmaceutically acceptable carrier. As used herein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Thetype of carrier can be selected based upon the intended route ofadministration. In various embodiments, the carrier is suitable forintravenous, intraperitoneal, subcutaneous, intramuscular, topical,transdermal or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethyelene glycol,and the like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the compounds can beadministered in a time release formulation, for example in a compositionwhich includes a slow release polymer, or in a fat pad described herein.The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, certain methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Depending on the route of administration, the compound may be coated ina material to protect it from the action of enzymes, acids and othernatural conditions which may inactivate the agent. For example, thecompound can be administered to a subject in an appropriate carrier ordiluent co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) andtrasylol. Liposonies include water-in-oil-in-water emulsions as well asconventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27).Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

The active agent in the composition (i.e., FFA1 inhibitor) preferably isformulated in the composition in a therapeutically effective amount. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result to thereby influence the therapeutic course of aparticular disease state. A therapeutically effective amount of anactive agent may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the agent toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the agent are outweighed by the therapeutically beneficial effects.In another embodiment, the active agent is formulated in the compositionin a prophylactically effective amount. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

The amount of active compound in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Exemplary dosages of compounds (e.g., FFA t inhibitor) of the inventioninclude e.g., about 0.0001% to 5%, about 0.0001% to 1%, about 0.0001% to0.1%, about 0.001% to 0.1%, about 0.005%-0.1%, about 0.01% to 0.1%,about 0.01% to 0.05% and about 0.05% to 0.1%.

The compound(s) of the invention can be administered in a manner thatprolongs the duration of the bioavailability of the compound(s),increases the duration of action of the compound(s) and the release timeframe of the compound by an amount selected from the group consisting ofat least 3 hours, at least 6 hours, at least 12 hours, at least 24hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 2 weeks, at least 3weeks, and at least a month, but at least some amount over that of thecompound(s) in the absence of the fat pad delivery system. Optionally,the duration of any or all of the preceding effects is extended by atleast 30 minutes, at least an hour, at least 2 hours, at least 3 hours,at least 6 hours, at least 12 hours, at least 24 hours, at least 48hours, at least 72 hours, at least 4 days, at least 5 days, at least 6days, at least 7 days, at least 2 weeks, at least 3 weeks or at least amonth.

A compound of the invention can be formulated into a pharmaceuticalcomposition wherein the compound is the only active agent therein.Alternatively, the pharmaceutical composition can contain additionalactive agents. For example, two or more compounds of the invention maybe used in combination. Moreover, a compound of the invention can becombined with one or more other agents that have modulatory effects oncancer.

Kits

The invention also includes kits that include a composition of theinvention, optionally also including a compound (e.g., a FFA1inhibitor), and instructions for use.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES Example 1: Materials and Methods Animals

All studies adhered to the NIH guide for the Care and Use of laboratoryanimals, the Association for Research in Vision and Ophthalmology (ARVO)Statement for the Use of Animals in Ophthalmic and Vision Research andwere approved by the Institutional Animal Care and Use Committee atBoston Children's Hospital. Vldlr knockout mice (Vldlr^(−/−); JacksonLab Stock: 002529) were crossed with wild type C57Bl/6 mice to obtainheterozygous breeders for littermate controlled experiments. Vldlr^(−/−)mice were also crossed with Ffar1 knockout mice (Ffar1^(−/−))¹ toultimately obtain Vldlr^(−/−)/Ffar1^(−/+) heterozygous breeders anddouble knockout mice (Vldlr^(−/−)/Ffar1^(−/−)). Pups weighing less than5 grams or more than 7 grams at postnatal day (P)16 were excluded(Stahl, A., et al. Am J Pathol 177, 2715-2723 (2010)). LittermateVldlr^(−/−) pups were treated from P8 to P15 with WY164363 (50 mg/kgonce daily, intraperitoneal; Sigma), GW9508 (14 μM, once dailyintraperitoneal; Cayman), TAK-875 (15 mg/kg twice daily, gavage;Selleckchem), medium chain triglyceride oil (MCT, 20 μL once dailygavage; Nestle) or corresponding vehicle and sacrificed at P16 toquantify retinal vascular lesions. Mice pups of both genders were used.

Quantification of Vascular Lesions

For quantification of outer retina vascular lesions, reminiscent ofretinal angiomatous proliferation (RAP) or macular telangiectasia(MacTel), mice were euthanized with a mixture of xylazine and ketamineand eyes were enucleated and fixed in 4% paraformaldehyde for 1 h atroom temperature. Retinas were dissected, carefully removing all hyaloidvessels, and stained overnight at room temperature with fluoresceinatedIsolectin B₄ (lectin) (Alexa Fluor 594-121413, Molecular Probes) in 1 mMCaCl₂ in PBS. Lectin-stained retinas were whole-mounted ontoSuperfrost/Plus microscope slides (Fisher Scientific) with thephotoreceptor side up and embedded in SlowFade Antifade reagent(invitrogen). For quantification of retinal lesions 20 images of eachwhole-mounted retina were obtained at 10× magnification on a ZeissAxioObserver.Z1 microscope and merged to form one image using AxioVision4.6.3.0 software. Vascular lesion counts were analyzed using theSWIFT_MACTEL method, an adaptation of the method used to measureneovascularization (SWIFT_NV) (Stahl, A., et al. Angiogenesis 12,297-301 (2009) in the oxygen induced retinopathy model.

SWIFT MACTEL

A set of macros were created that were developed to run on ImageJplatform (National Institutes of Health, http://imagej.nih.gov/ij/). Inbrief, SWIFT_MACTEL isolates the red channel from a lectin-stainedretinal whole mount, divides the image into four quadrants and removesbackground fluorescence to allow for the neovascularization (NV)structures to stand out clearly against the background fluorescence ofnormal vessels. Using a slide bar to either increase or decrease aparticular quadrant's fluorescence threshold, the SWIFT_MACTEL userdesignates a threshold that marks NV structures but not normal vesselsto each quadrant. After setting the appropriate threshold, artifactslike cellular debris or hyperfluorescent retinal edges can be manuallyremoved and excluded from quantification. SWIFT_MACTEL then analyzes allpixels in the image that lie above the chosen intensity threshold andare part of an object that has a minimum size of 100 pixels. By settingthis cut-off in object size, small artifacts like vessel branch pointsare automatically removed. After measuring all four quadrants,SWIFT_MACTEL creates a composite from all four NV quadrants andcalculates the total NV pixel number. Results from the SWIFT_NV methodhave been found to correlate well with results from the established handmeasurement protocols (R²=0.9372) and show robust intra-individual(R²=0.9376) and inter-individual (R²=0.9424) reproducibility (Stahl, A.,et al. Angiogenesis 12, 297-301 (2009)). n is number of eyes quantified.

Scanning Electron Microscopy and 3D Retinal Reconstruction

Tissue was processed for serial block face scanning electron microscopy(SEM) using an adapted version of a protocol established by Deerinck etal. 2010 (T. Deerinck, et al., Microscopy and Microanalysis 16, no. S2(2010)). Whole eyes were isolated and fixed in Karnovsky's fixative. Thecornea and lens were removed and the tissue further fixed in tannic acidovernight. Heavy metal infiltration was then undertaken; tissue wasincubated in 1.5% potassium ferrocyanide, 0.5% osmium tetroxide incacodylate buffer, followed by thiocarbohydrazide treatment and a secondexposure to 1% osmium incubation. Walton's lead aspartate exposure wasnot carried out, so they were finished with 1% uranyl acetate incubationfollowed by dehydration to propylene oxide and embedded in Durcupan ACMresin. The tissue was serially sectioned and imaged using the Gatan3VIEW serial block face imaging system (Gatan, Abingdon, UK) fitted to aZeiss Sigma variable pressure field emission scanning electronmicroscope (Zeiss, Cambridge, UK). Data was collected and used in AmiraSoftware (FEI, Oregon, USA) in order to reconstruct the 3D images. Usingthe same software, photoreceptor mitochondrial volume was estimated forWT mice, around the lesions in Vldlr^(−/−) mice, and away from thelesion in Vldlr^(−/−) mice.

ATP Measurement

ATP was measured using a kit per instruction manual (Molecular Probes,A22066). Briefly, a standard reaction solution was made from thefollowing components: dH₂O, 20× Reaction Buffer, DTT (0.1 M),D-luciferin (10 mM), firefly luciferase stock solution (5 mg/mL).Low-concentration ATP standard solutions were prepared by diluting ATPsolution (5 mM) in dH₂O. A standard curve was generated by subtractingthe background luminescence of the standard reaction solution fromluminescence readings for a series of dilute ATP standard solutions.Luminescence measurements were taken for ATP-containing samples and theamount of ATP in experimental samples were calculated from the standardcurve.

Oxygen Consumption and Extracellular Acidification Rates

All oxygen consumption rates were measured using a Seahorse XFe96 FluxAnalyzer®. Whole retinas were isolated and 1 mm punch biopsies wereloaded into the 96 well plate. Retinal punches were incubated in assaymedia (DMEM 5030 media supplemented with 12 mM glucose, 10 mM HEPES and26 mM NaHCO₃) to measure oxygen consumption rates (OCR) andextracellular acidification rates (ECAR). Photoreceptor (661W) cells,were incubated in their assay media (DMEM 5030, 12 mM glucose, 10 mMHEPES) one hour prior to measurements. Fatty acid oxidation rates weredetermined by treating tissues or cells with Etomoxir (40 μM; Sigma) 40min prior to analysis and then providing BSA (control) or BSA/Palmitateconjugate (Seahorse). Glucose oxidation rates were measured afterinjection of 2-deoxyglucose (2-DG, 100 mM; Sigma) or media controlduring data acquisition. To determine the maximal fatty acid or glucoseoxidative capacity, the nonmitochondrial respiration (rate afterinjection of 2 μM Rotenone and 2 μM Antimycin A) was subtracted from theoxygen consumption rate after injection of 0.5 μM carbonylcyanide-ptrifluoromethoxyphenylhydrazone (FCCP).

Glucose and Lactate Measurements

Whole retinas and photoreceptors (661W) were incubated in assay media(DMEM 5030, 12 mM glucose, 10 mM HEPES) for 6 and 48 hours respectively.Media was collected, spun briefly (13 g) to remove cellular debris, andglucose and lactate levels measured using a Yellow Springs Instrument(YSI) 2950 were compared to control media that was not exposed to tissueor cells. To determine the conversion of glucose to lactate (theglycolytic rate), lactate production was divided by glucose uptake.

Retinal Lipid Uptake

Retinal long-chain fatty acid uptake was compared for wild type andVldlr^(−/−) mice gavaged with 0.1 mg of4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-hexadecanoicacid (BODIPY FL C16; Molecular Probes). Mice were euthanized 2 h laterand the eyes were enucleated, embedded in OCT and cryo-sectioned (10 μm)for immediate imaging by fluorescence microscopy. Retinal FA uptake wasquantitated using ¹⁴C-labeled 2-bromopalmitate tracer injected twicedaily (i.p.; 0.5 uCi per dose) from P9 to P12; total administeredradioactivity was normalized for mouse body weight. Retinas were thendissected and homogenized in Ultima Gold liquid scintillation cocktail(PerkinElmer) and beta-counted (¹⁴C DPM) using Tri-Carb 2900TRinstrument (PerkinElmer), correcting for background scintillation.

Plasma Triglycerides (TG) and Fatty Acid (FA) Analysis

Plasma was isolated from WT and Vldlr^(−/−) mice by centrifugation (2000g×20 min; 4° C.) of whole blood in EDTA-coated tubes. TGs weredetermined using the RANDOX TRIGS kit (TR210) per instruction manual.FAs in whole plasma were assayed as described previously (Spahis, S., etal. Prostaglandins Leukor Essent Fatty Acids 99, 25-34 (2015)). Briefly,each sample was subjected to direct transesterification and theninjected into a gas chromatograph using the Agilent GC AutoSamplersystem (7890A). FAs were identified by comparison with the expectedretention times of known standards and then analyzed with OpenLABSoftware Suite (Agilent).

β-Oxidation Metabolite Quantification

Acylcarnitine metabolites were extracted from WT and Vldlr^(−/−) (P12)flash frozen retinas using ice-cold methanol. Samples were sonicated,centrifuged and the supernatant was transferred to a fresh tube fornitrogen evaporation. When dry, butanolysis was performed (butanol-HCl.55° C. for 20 min) prior to reconstitution in mobile phase (ACN:H₂O80:20, formic acid 0.05%). Samples were analyzed by liquidchromatography followed by tandem mass spectrophotometry (LC/MS/MS,Alliance 2795 LC and Quattro micro, Waters Corp). Data were recorded inpositive electrospray ionization and analyzed with Neolynx (WatersCorp).

Retinal Glucose Uptake

Positron emission tomography (PET) imaging studies were performed on WT,Vldlr^(−/−), Vldlr^(−/−)/Ffar1^(−/−) and Ffar1^(−/−) mice (P16; Focus120 high-resolution, Siemens), followed by micro CAT imaging (MicroCATII scanner, Siemens). Fluorine-18 fluorodeoxyglucose (¹⁸F-FDG) wasadministered by intraperitoneal injection (i.p.) to obtain nontoxicradioactivity levels (3.7 and 37 MBq; or 0.1 to 1.0 mCi). Actualadministered activity was determined using a dose calibrator to measureactivity in the syringe before and after the injection. Images wereacquired 60 minutes after injection to ensure radiotracer uptake. Micewere fasted for 6 hours prior to imaging, kept in darkness andanesthetized by inhalation of isoflurane (2-4%) through a nose cone forthe duration of the procedure. Animals were imaged in a head first,prone position, and placed on a heating pad to maintain appropriate bodytemperature. Upon completion of imaging, mice were euthanized andretinas were dissected for precise ¹⁸F-FDG retinal activity,quantification by gamma counter, and corrected for decay. WT andVldlr^(−/−) mice pups were also injected with trace amounts of³H-2-deoxyglucose (0.5 μCi daily, i.p.) and treated with GW9508 orvehicle (14 μM daily, i.p.) for 5 days (P7-P12). Retinas were collectedand homogenized in a scintillation cocktail (Ecolite+, MP biomedicals)and betacounts measured using the LS6500 Multipurpose ScintillationCounter (Beckman).

Laser-Capture Microdissection

Eyes were embedded in OCT and flash frozen immediately followingenucleation. Eyes were cryosectioned under RNase free conditions into 10μm sections, and collected on RNase-free polyethylene naphthalate glassslides (11505189, Leica). Sections were stained for lectin (1:50 in 1 mMCaCl₂) and dehydrated with 70%, 90% and 100% ethanol washes. Retinalvessels and layers were microdissected with a Leica LMD 6000 system(Leica Microsystems) and collected directly into RNA stabilizing bufferfrom the RNeasy Micro kit (Qiagen, Chatsworth, CA). RNA was extractedfrom microdissected tissues using the RNeasy Kit as described above(Qiagen), and real-time PCR was performed with the generated cDNA.

Reverse Transcription and Quantitative Real-Time PCR Analysis

RNA samples from cell culture, whole retina or laser-captured neovesselsand layers were treated with DNase 1 (Qiagen, Chatsworth, CA) to removeany contaminating genomic DNA. The DNase-treated RNA was then convertedinto cDNA using reverse transcriptase (Invitrogen). PCR primers fortarget genes and the control gene, cyclophilin A, were designed usingPrimer Bank and NCBI Primer Blast software. Quantitative analysis ofgene expression was generated using an ABI Prism 7700 Sequence DetectionSystem with the SYBR Green Master mix kit and gene expression wascalculated relative to cyclophilin A using the ΔcT method.

Primer sequences Gene Forward (5′-3′) Reverse (5′-3′) Cptla CATGTCAAGCTGGTAGGAGA CAGAGGAAGA GCAGCACCTT Cyclophillin CAGACGCCAC TGTCTTTGGA ATGTCGCTTT ACTTTGTCTG CAA Glut1 CAGTTCGGCT GCCCCCGACA ATAACACTGGGAGAAGATG TG Ffar4 CTTTCTTCTC GATGAGCCCC (GPR120) GGATGTCAAG AGAACGGTGGG Ffar1 CCTTCGCTCT GGCTAACAAG (GPR40) CTATGTATCT TTCAATGGAA GCC AGCFfar3 TTCTGAGCGT AGACTACACT (GPR41) GGCCTATCCA GACCAGACCA G Ffar2TGCTACGAGA CACACGAAGC (GPR43) ACTTCACCCA GCCAATAACA A G GPR84 CCAGCGAGGGGCTTCTGACG GATTTCATCT AATCACCTTC G CA Pkm2 ATCGGGCGAT AGAGGGCCATGCAACCGAGC CAAGGTACAG GCA Pparα AGAGCCCCAT ACTGGTAGTC CTGTCCTCTCTGCAAAACCA AA Pparβ/δ TCCATCGTCA ACTTGGGCTC ACAAAGACGG AATGATGTCA G CPparγ TCGCTGATGC GAGAGGTCCA ACTGCCTATG CAGAGCTGAT T VE- ATTGGCCTGTCACAGTGGGG Cadherin GTTTTCGCAC TCATCTGCAT Vldlr TCTCTTGCTC CTTACAACTGTTAGTGATGG ATATTGCTGG G

Expression Array

Illumina mouse gene microarray analysis of WT and Vldlr^(−/−) retinaswas performed in biological triplicate (Mouse-WG6 expression BeadChip,Illumina). The chip contains 45,000 probe sets representing 34,000genes. Microarray studies, from cDNA synthesis to raw data normalizationwere performed by the Molecular Genetics Core Facility at BostonChildren's Hospital. Briefly, total RNA (1 μg each) was reversetranscribed, followed by a single in vitro transcription amplificationto incorporate biotin-labeled nucleotide, and subsequent hybridizationand staining with strepatavidin-Cy3 according to the manufacturer'sinstructions. The chip was scanned with Illumina BeadArray Reader tomeasure the signal intensity by the labeled target. Raw data wereanalyzed with the microarray software (Bead Studio Gene Expressionversion 3.4.0) for quality control, background analysis andnormalization with rank invariant algorithm. Normalized data was furtheranalyzed for comparative molecular and cellular pathway regulation usingIngenuity Pathway Analysis (P=0.05 and delta of 0.19; Quiagen) (Calvano,S. E., et al. Nature 437, 1032-1037 (2005)).

Immunohistochemistry

For whole mount immunohistochemistry, eyes were enucleated and fixed in4% paraformaldehyde at room temperature for 1 h. The retina was isolatedand stained for retinal vasculature and lesions with fluoresceinatedIsolectin B4 (Alexa Fluor-594 in PBS with CaCl₂ 1 mM, 121413, MolecularProbes) overnight at room temperature (RT). Retinas were visualizedusing a 5× objective with a Zeiss AxioObserver.Z1 microscope, and imagedwith a Zeiss AxioCam MRm operated by AxioVision software (version4.6.3.0). Whole mounts were also fixed and permeabilized in coldmethanol (20 min at −20° C.), blocked in 3% bovine serum albumin and0.1% Triton X-100, stained with Isolectin B₄ to visualize vessels (asabove) and/or with primary antibodies against HIF1α (1:100 in TBS,NB00-134, Novus), VEGF (1:100, RB-222, Thermo Scientific), IBA-1 (1:200,CP290A, Biocare Medical UK) and blue opsin cone (1:100, sc-14365, SantaCruz) overnight at 4° C., followed by secondary antibody staining (1hour at RT; AlexaFluor 1:1000, Invitrogen). Flatmounts (andcross-section) were imaged with confocal microscopy (Leica TCS SP2 AOBS)and z-stacks were 3D reconstructed using Volocity software (Perk Elmer).

Western Blot and ELISA

Retinal samples were obtained as described above. Retinal lysate (20 μg)from three different animals or endothelial cells lysate (10 μg) wereloaded on an SDS-PAGE gel and electroblotted onto a PVDF membrane. Afterblocking, the membranes were incubated with antibodies against β-Actin(Sigma, A 1978), Hif1α (Novus, NBI00-134), and Glut1 (Novus, NB300-666and Abcam, Ab652) overnight (1:1000 each). After washing, membranes wereincubated with 1:10,000 horseradish peroxidase-conjugated anti-rabbit oranti-mouse secondary antibodies (Amersham, NA931V and NA934V) for onehour at room temperature. Densitometry was analyzed using Image. RetinalVegfa concentration was measured by ELISA (as per manual, MMV00, R&DSystems) and normalized by doing a Bradford to measure the total cellprotein content of each samples.

Metabolite Profiling

Metabolites of rapidly dissected WT and Vldlr^(−/−) retinas (flashfrozen less than a minute from euthanasia; 15-16 biological replicates)were homogenized in 80% methanol (8 μL/mg of tissue) containing theinternal standards inosine-¹⁵N₄, thymine-d₄, and glycocholate-d₄(Cambridge Isotope Laboratories) using a TissueLyser II (Qiagen) beadmill for 4 minutes at 20 Hz. Samples were centrifuged (9,000 g, 10 min,4° C.) to pellet debris and supernatants were analyzed using two liquidchromatography tandem mass spectrometry (LC-MS) methods to measure polarmetabolites as described previously (Jain, M., et al. Science 336,1040-1044 (2012), and Townsend, M. K., et al Clin Chem 59,1657-1667(2013). Briefly, negative ionization mode multiple reactionmode (MRM) data were acquired using an ACQUITY UPLC (Waters) coupled toa 5500 QTRAP triple quadrupole mass spectrometer (AB SCIEX). Thesupernatants were injected directly onto a 150×2.0 mm Luna NH₂ column(Phenomenex) that was eluted at a flow rate of 400 μL/min with initialconditions of 10% mobile phase A [20 mM ammonium acetate and 20 mMammonium hydroxide (Sigma-Aldrich) in water (VWR)] and 90% mobile phaseB [10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol (VWR)]followed by a 10 min linear gradient to 100% mobile phase A. The ionspray voltage was −4.5 kV and the source temperature was 500° C.Positive ionization mode MRM data were acquired using a 4000 QTRAPtriple quadrupole mass spectrometer (AB SCIEX) coupled to an 1100 Seriespump (Agilent) and an HTS PAL autosampler (Leap Technologies). Cellextracts (10 μL) were diluted using 40 μL of 74.9:24.9:0.2 (v/v/v)acetonitrile/methanol/formic acids containing stable isotope-labeledinternal standards [0.2 ng/μL valine-d8, Isotec; and 0.2 ng/μLphenylalanine-d8 (Cambridge Isotope Laboratories)] and were injectedonto a 150×2.1 mm Atlantis HILIC column (Waters). The column was elutedisocratically at a flow rate of 250 μL/min with 5% mobile phase A (10 mMammonium formate and 0.1% formic acid in water) for 1 minute followed bya linear gradient to 40% mobile phase B (acetonitrile with 0.1% formicacid) over 10 minutes. The ion spray voltage was 4.5 kV and the sourcetemperature was 450° C. Raw data were processed using MultiQuant 2.1 (ABSCIEX) for automated peak integration and metabolite peaks were manuallyreviewed for quality of integration and compared against known standardsto confirm identity.

Photoreceptor (661W) Cell Culture

Cone photoreceptor cells (al-Ubaidi, M. R., J Cell Biol 119, 1681-1687(1992), and Tan, E., et al. Invest Ophthalmol Vis Sci 45, 764-768(2004)) (661 W; from Dr. Al-Ubaidi) were cultured as monolayers at 37°C., 5% CO₂ in a humidified atmosphere in DMEM with FBS 10% supplementedwith hydrocortisone (20 μg/500 mL, H-2270, Sigma), Progesterone (20μg/500 mL, P-8783, Sigma), Putrescine (0.016 g/500 mL, P-7505, Sigma)and β-mercaptoethanol (20 μL/500 mL, M-6250, Sigma). Equal number of661W cells (0.3×106) were plated in 6-well dishes and cultured to 80%confluence. Cells were washed twice with PBS, starved for 4 hours (abovemedium without FBS) then stimulated with GW9508 (14 μM, Cayman) orvehicle. Photoreceptors were then collected 8 hours post-treatment forHif1α protein expression (see Western blot); while their medium wascollected at 12 hours for Vegfa quantification by ELISA (as per manual,MMV00, R&D Systems). Vegfa concentration was normalized for the numberof cells per well, by doing a Bradford to measure the total cell proteincontent of each well.

Preparation of AAV2-RKshVldlr Vector and AAV2 Virus

Three independent shRNAs against mouse Vldlr were designed using apublished algorithm (Park, Y. K., et al. Nucleic acids research 36,W97-103 (2008)). The template oligonucleotides contained miR-30microRNA, miR-30 loop and Vldlr shRNA including the sense and theantisense were synthesized (Invitrogen). DNA fragments were amplified,purified, digested and inserted into modified CAG-GFP-miR30 vector(provided by Dr. Zhiqiang Lin and Dr. William T. Pu at Boston Children'sHospital) according to a previous report (Grieger, J. C., et al., Natureprotocols 1, 1412-1428 (2006)) and CAG promoter was replaced withrhodopsin kinase (RK) promoter (Khani, S. C., et al. Investigativeophthalmology & visual science 48, 3954-3961 (2007)) that was clonedfrom pAAVRK-GFP (provided by Dr. Connie Cepko and Dr. Tiansen Li). TheVldlr knock down efficiency was tested in pup retinas. Recombinant AAV2vectors were produced as previously described (Vandenberghe, L. H., etal. Human gene therapy 21, 1251-1257 (2010)). Briefly, AAV vector,rep/cap packaging plasmid, and adenoviral helper plasmid were mixed withpolyethylenimine and transfected into HEK293T cells (CRL-11268, ATCC).Seventy-two hours after transfection, cells were harvested and the cellpellet was resuspended in virus buffer, followed by 3 cycles offreeze-thaw, and homogenized. Cell debris was pelleted at 5,000 g for 20minutes, and the supernatant was run on an iodixanol gradient. RecoveredAAV vectors were washed 3 times with PBS using Amicon 100K columns (EMDMillipore). Real-time PCR was used to determine genome titers of therecombinant AAV. This protocol also was used to prepare a controlAAV2-shControl. Viruses were diluted to various concentrations to testinfection, and a concentration of approximately 2×10¹² gc/mL was usedfor the experiments. The sequences of the mouse Vldlr siRNAs are asfollows: shVldlr #1 (5′-3′), GGA AAG TTC AAG TGC AGA AGC G; shVldlr #2(5′-3′), GGA ATG CCA TAT CAA CGA ATG C; shVldlr #3 (5′-3′), GGG ATC TGCAGT CAA ATT TGT A; Scramble shRNA control (5′-3′), GAT TTA AGA CAA GCGTAT AAC A.

Human Samples and Vitrectomy

The study conforms to the tenets of the Declaration of Helsinki, andapproval of the human clinical protocol and informed consent wereobtained from the Maisonneuve-Rosemont Hospital (HMR) ethics committee(Ref. CER: 10059). All patients previously diagnosed with AMD werefollowed and surgery was performed by a single vitreoretinal surgeon(F.A.R.). Vitreous samples were frozen on dry ice immediately afterbiopsy and stored (−80° C.). VEGFA ELISAs were performed according tomanufacturer's instructions (DVE00, R&D Systems).

Statistical Analysis

A Student's t-test was used, and ANOVA with Dunnet, Bonferroni or Tukeypost-hoc analysis (see Table 1), to compare different groups; p<0.05 wasconsidered statistically different. D'Agostino-Pearson orKolmogorov-Smirnov (KS) normality test were used to confirm normaldistribution. Data with non-Gaussian distribution was analyzed using aMann Whitney test (non-parametric, two groups). Animals were notrandomized but quantifications were blinded when possible. Allexperiments were repeated at least 3 times. Values more than 2 standarddeviations from the mean were considered outliers and were excluded.Sample size was estimated to detect a difference of 20% with a power of80% (1-β) and a of 0.05 in accordance with the ‘Guidelines for the Useof Animals in Neuroscience’ (2003). Results are presented as mean±SEM.*P<0.05, **P<0.01, ***P<0.001.

Description of Statistical Analysis

Detailed description of n, difference in variance, statistical test andP values for each figure panel (Table 1):

Difference in FIG. & Variance PANEL Groups n (F test, P value)Statistical Test P value FIG. 1 a Schematic b 5 retinas/time pointDescriptive c Control 28 retinas F = 2.848, P = 0.1034 Unpairedtwo-tailed 0.0031 (Dark/Light) 10 retinas Student t-test Dark d WT 23photoreceptors F = 21.91 P < 0.0001 Unpaired two-tailed <0.0001Vldlr^(−/−) 23 photoreceptors Student t-test with Welch's correction eWT 4 retinas (littermate) F = 3.032, P = 0.2635 Unpaired two-tailed0.0026 Vldlr^(−/−) 6 retinas (littermate) Student t-test FIG. 2 a, b Ctl(BSA) 7 retinas One-way ANOVA, Tukey's Multiple Ctl(BSA) vs Palmitate 6retinas F = 9.783 P = 0.0002 Comparison Test Palmitate: <0.01 Ctl +Etomoxir 7 retinas Palmitate vs Palmitate + 8 retinas BSA + Eto: <0.001Etomoxir Palmitate vs Palm + Eto: <0.01 c WT 7 plasmas (from 2 F =22.56, P = 0.0011 Unpaired two-tailed <0.0001 Vldlr^(−/−) animals each)Student t-test with 13 plasmas (from 2 Welch's correction animals each)d WT 3 animals (×2 β-Oxidation Metabolite Vldlr^(−/−) pooled retinas)per Array group (littermate) e WT 3 animals (×2 pooled F = 2.802, P =0.5260 Unpaired two-tailed 0.0108 left Vldlr^(−/−) retinas) per groupStudent t-test e WT 3 animals (×2 F = 1.798, P = 0.7147 Unpairedtwo-tailed 0.0014 right Vldlr^(−/−) pooled retinas) per Student t-testgroup (littermate) f WT 3 animals (×2 F = 1.079, P = 0.9620 Unpairedtwo-tailed 0.0052 left Vldlr^(−/−) pooled retinas) per Student t-testgroup (littermate) f WT 3 animals (×2 One-way ANOVA, Tukey's MultipleONL WT vs right Vldlr^(−/−) pooled retinas) per F = 15.09 P < 0.0001Comparison Test Vldlr^(−/−): <0.001 group (littermate) g WT 22 retinas F= 1.225, P = 0.7492 Unpaired two-tailed 0.0116 Vldlr^(−/−) 12 retinasStudent t-test h WT 9 retinas F = 11.61, P = 0.0005 Unpaired two-tailed0.0119 left Vldlr^(−/−) 12 retinas Student t-test with Welch'scorrection h WT 3 animals (6 One-way ANOVA, Tukey's Multiple ONL - WT vsright Vldlr^(−/−) retinas)/group F = 60.63, P < 0.0001 Comparison TestVldlr^(−/−): <0.001 Littermate INL - WT vs controlled Vldlr^(−/−):<0.001 i WT 6 retinas F = 1.496, P = 0.6691 Unpaired two-tailed 0.0300Vldlr^(−/−) 6 retinas Student t-test FIG. 3 a Schematic b WT 3 animals(6 retinas) One way ANOVA Dunnett's Multiple Ffar1 vs all Vldlr^(−/−) 3animals (6 retinas) F = 91.38, P < 0.0001 Comparison Test other One wayANOVA Ffar: <0.001 F = 816.5, P < 0.0001 c WT 3 animals (6 retinas)One-way ANOVA, Dunnett's Multiple ONL vs other Vldlr^(−/−) 3 animals (6retinas) F = 209.5, P < 0.0001 Comparison Test layers: <0.001 One-wayANOVA, F = 82.14, P < 0.0001 d Ctl 5 retinas One-way ANOVA, Tukey'sMultiple WT-Ctl vs WT GW 8 retinas F = 20.03, P < 0.0001 Comparison TestWT-GW: <0.001 d Ctl 9 retinas WT-Ctl vs Vldlr^(−/−) GW 16 retinasVldlr^(−/−)-Ctl: <0.01 WT-Ctl vs Vldlr^(−/−)-GW: <0.001 Vldlr^(−/−)-Ctlvs Vldlr^(−/−)-GW: <0.01 e Ctl 12 retinas F = 5.570, P = 0.0083 Unpairedtwo-tailed <0.0001 GW 12 retinas Student t-test with Welch's correctionf Ctl 7 retinas F = 4.719, P = 0.0708 Unpaired two-tailed 0.0002 GW 11retinas Student t-test g WT 4 retinas One-way ANOVA, Dunnett's MultipleVldlr^(−/−) vs other Vldlr^(−/−) 4 retinas F = 11.75, P = 0.0031Comparison Test groups: <0.05 Vldlr^(−/−)/Ffar1^(−/−) 4 retinas h WT 10retinas One-way ANOVA, Dunnett's Multiple Vldlr^(−/−) vs otherVldlr^(−/−) 9 retinas F = 4.89, P = 0.0161 Comparison Test groups: <0.05Vldlr^(−/−)/Ffar1^(−/−) 9 retinas i Vldlr^(−/−) 10 retinas F = 1.075, P= 0.9159 Unpaired two-tailed 0.0153 Vldlr^(−/−)/Ffar1^(−/−) 10 retinasStudent t-test FIG. 4 a Schematic b WT 15 animals (12 × 2 non Gaussiantwo-tailed Mann 0.0032 Vldlr^(−/−) pooled retinas) distribution Whitneytest 12 animals (15 × 2 pooled retinas) c WT 3 animals (2 F = 2.362, P =0.5949 Unpaired two-tailed 0.0094 Vldlr^(−/−) pooled retinas) Studentt-test 3 animals (2 pooled retinas) d WT 11 animals (12 × 2 F = 2.622, P= 0.0974 Unpaired two-tailed 0.0069 Vldlr^(−/−) pooled retinas) Studentt-test 15 animals (15 × 2 pooled retinas) e WT 15 animals (12 × 2 nonGaussian two-tailed Mann 0.0004 Vldlr^(−/−) pooled retinas) distributionWhitney test 12 animals (15 × 2 pooled retinas) f WT 3 retinas One-wayANOVA, Dunnett's Multiple WT vs Vldlr^(−/−) 3 retinas F = 4.87, P =0.0554 Comparison Test Vldlr^(−/−): <0.05 Vldlr^(−/−)/Ffar1^(−/−) 3retinas g Vldlr^(−/−) 3 retinas descriptive h WT 6 retinas One-wayANOVA, Tukey's Multiple WT vs Vldlr^(−/−) 6 retinas F = 21.68, P <0.0001 Comparison Test Vldlr^(−/−): <0.001 Vldlr^(−/−)/Ffar1^(−/−) 6retinas Vldlr^(−/−) vs Vldlr^(−/−)/ Ffar1^(−/−): <0.01 i 3 retinasdescriptive j Ctl 8 vitreous samples One-way ANOVA, Dunnett's MultipleCtl vs other CNV 7 vitreous samples F = 4.972, P = 0.0221 ComparisonTest groups <0.05 RAP 3 vitreous samples FIG. 5 a WT 4 retinas One-wayANOVA, Dunnett's Multiple WT vs Vldlr^(−/−) (het) 5 retinas F = 44.01, P< 0.0001 Comparison Test Vldlr^(−/−): <0.01 Vldlr^(−/−) 5 retinas WT vsVldlr^(−/−): <0.001 Vldlr^(−/−) vs Vldlr^(−/−): <0.01 b WT 7 plasmas (2F = 6.855, P = 0.0229 Unpaired two-tailed 0.5596 (n.s.) Vldlr^(−/−)(het) pooled mice each) Student t-test with 8 plasmas (2 Weich'scorrection pooled mice each) c WT 7 plasmas (2 F = 3.398, P = 0.1346Unpaired two-tailed 0.6969 (n.s.) Vldlr^(−/−) (het) pooled mice each)Student t-test 8 plasmas (2 pooled mice each) d, e Schematics f, g 5retinas/time point Descriptive h shCtl 8 retinas One-way ANOVA,Dunnert's Multiple shCtl vs shRNA Vldlr - 1 12 retinas F = 7.227, P =0.0010 Comparison Test shVldlr 1: <0.05 shRNA Vldlr - 2 4 retinas shCtlvs shRNA Vldlr - 3 8 retinas shVldlr 2: <0.01 shCtl vs shVldlr 3: <0.001i 5 retinas/time point Descriptive FIG. 6 a Schematic b, c Palmitate 6retinas non Gaussian two-tailed Mann 0.0027 Palmitae + 8 retinasdistribution Whitney test Etomoxir d, e Glucose 8 retinas F = 1.512, P =0.5989 Unpaired two-tailed 0.0019 Glucose + 2-DG 8 retinas Studentt-test f Glucose 8 retinas non Gaussian two-tailed Mann 0.8518 (n.s.)Palmitate 6 retinas distribution Whitney test g Glucose 4 retinasdescriptive Lactate 4 retinas h WT - Ctl 15 retinas F = 3.632, P =0.0229 Unpaired two-tailed 0.0035 left WT - Palmitate 14 retinas Studentt-test with Welch's correction h Vldlr^(−/−) - Ctl 10 retinas F = 1.345,P = 0.6194 Unpaired two-tailed 0.5758 (n.s.) right Vldlr^(−/−) - 13retinas Student t-test Palmitate FIG. 7 a WT 3 retinas One-way ANOVA,Tukey's Multiple ONL vs all F = 130.6, P < 0.0001 Comparison Testlayers: <0.001 RPE vs GC layer: <0.05 b WT 3 retinas descriptiveVldlr^(−/−) 3 retinas c WT 16 retinas F = 1.051, P = 0.9540 Unpairedtwo-tailed 0.0003 Vldlr^(−/−) 12 retinas Student t-test d WT 7 plasmas(2 F = 10.37, P = 0.0092 Unpaired two-tailed <0.0001 Vldlr^(−/−) pooledmice each) Student t-test with 13 plasmas (2 Welch's correction pooledmice each) e WT 7 plasmas (2 non Gaussian two-tailed Mann 0.1322 C8Vldlr^(−/−) pooled mice each) distribution Whitney test 13 plasmas (2pooled mice each) C10 WT 7 plasma (2 non Gaussian two-tailed Mann 0.0012Vldlr^(−/−) pooled mice each) distribution Whitney test 13 plasmas (2pooled mice each) C12 WT 7 plasmas (2 F = 7.974, P = 0.0184 Unpairedtwo-tailed <0.0001 Vldlr^(−/−) pooled mice each) Student t-test with 13plasmas (2 Welch's correction pooled mice each) C14 WT 7 plasmas (2 F =12.71, P = 0.0053 Unpaired two-tailed <0.0001 Vldlr^(−/−) pooled miceeach) Student t-test with 13 plasmas (2 Welch's correction pooled miceeach) C16 WT 7 plasmas (2 F = 22.56, P = 0.0011 Unpaired two-tailed<0.0001 Vldlr^(−/−) pooled mice each) Student t-test with 13 plasmas (2Welch's correction pooled mice each) C18 WT 7 plasmas (2 F = 25.57, P =0.0007 Unpaired two-tailed <0.0001 Vldlr^(−/−) pooled mice each) Studentt-test with 13 plasmas (2 Welch's correction pooled mice each) C18:1n9WT 7 plasmas (2 F = 12.89, P = 0.0051 Unpaired two-tailed <0.0001Vldlr^(−/−) pooled mice each) Student t-test with 13 plasmas (2 Welch'scorrection pooled mice each) C18:2n6 WT 7 plasmas (2 F = 23.96, P =0.0009 Unpaired two-tailed <0.0001 Vldlr^(−/−) pooled mice each) Studentt-test with 13 plasmas (2 Welch's correction pooled mice each) C20:4n6WT 7 plasmas (2 F = 13.54, P = 0.0044 Unpaired two-tailed <0.0001Vldlr^(−/−) pooled mice each) Student t-test with 13 plasmas (2 Welch'scorrection pooled mice each) C22:6n3 WT 7 plasmas (2 F = 5.334, P =0.0507 Unpaired two-tailed <0.0001 Vldlr^(−/−) pooled mice each) Studentt-test 13 plasmas (2 pooled mice each) FIG. 8 a WT 3 animals (×2 F =427.2, P = 0.0047 Unpaired two-tailed 0.0079 Vldlr^(−/−) pooled retinas)per Student t-test with group (littermate) Welch's correction b WT 3animals (×2 One-way ANOVA, Tukey's Multiple WT ONL vs Vldlr^(−/−) pooledretinas) per F = 19.02, P < 0.0001 Comparison Test all other group(littermate) layers: <0.001 c Ctl 10 retinas F = 2.2, P = 0.218 Unpairedtwo-tailed 0.0429 WY16463 12 retinas Student t-test FIG. 9 a, b Veh -Ctl 5 retinas One-way ANOVA, Tukey's Multiple Ctl + Veh vs Veh -Palmitate 5 retinas F = 55.54, P < 0.0001 Comparison Test Palmitate +Etomoxir - Ctl 6 retinas veh: <0.001 Etomoxir - 6 retinas Palmitate +Veh Palmitate vs Ctl or Palm + Eto: <0.001 c, d Veh - Ctl 5 retinasOne-way ANOVA, Tukey's Multiple Ctl + Veh vs Veh - Palmitate 5 retinas F= 89.67, P < 0.0001 Comparison Test Palmitate + PPARa agonist - 6retinas veh: <0.001 Ctl Palmitate + PPARa agonist PPARa + 5 retinas vsall Palmitate groups: <0.001 e, f Veh - Ctl 5 retinas non GaussianKruskal-Wallis with n.s. Veh - Palmitate 6 retinas distribution Dunn'sMultiple PPARa - Ctl 6 retinas Comparison Test PPARa + 4 retinasPalmitate FIG. 10 a WT 3 animals (×2 Ingenuity array Vldlr^(−/−) pooledretinas) per pathway analysis group (littermate) b WT 6 retinas F =52.4, P = 0.0019 Unpaired two-tailed 0.0215 Vldlr^(−/−) 5 retinasStudent t-test with Welch's correction c WT 4 retinas F = 1.262, P =0.8006 Unpaired two-tailed 0.8403 (n.s.) Vldlr^(−/−) 3 retinas Studentt-test WT 4 retinas F = 5.733, P = 0.1889 Unpaired two-tailed 0.4517(n.s.) Vldlr^(−/−) 3 retinas Student t-test FIG. 11 a WT - Ctl 11retinas F = 3.055, P = 0.0927 Unpaired two-tailed 0.0284 WT - GW 11retinas Student t-test Vldlr^(−/−) - Ctl 16 retinas F = 1.430, P =0.5031 Unpaired two-tailed 0.0142 Vldlr^(−/−) - GW 14 retinas Studentt-test b WT 17 retinas One-way ANOVA, Dunnett's Multiple WT vsVldlr^(−/−) 6 retinas F = 6.613, P < 0.0008 Comparison Test Vldlr^(−/−):<0.001 Vldlr^(−/−)/Ffar1^(−/−) 10 retinas Ffar1^(−/−) 16 retinas c WT 9retinas One-way ANOVA, Dunnett's Multiple WT vs Vldlr^(−/−) 9 retinas F= 4.762, P < 0.01 Comparison Test Vldlr^(−/−): <0.01Vldlr^(−/−)/Ffar1^(−/−) 6 retinas Ffar1^(−/−) 3 retinas d siNT 3experiments F = 12.4, P = 0.1493 Unpaired two-tailed 0.0025 siFfar1Student t-test e siNT - Ctl 3 experiments One-way ANOVA, Bonferroni'sMultiple <0.001 siNT - GW F = 38.99, P < 0.0001 Comparison siFfar1 - Ctl3 experiments Bonferroni's Multiple ns siFfar1 - GW Comparison f Ctl 3retinas F = 2.438, P = 0.5818 Unpaired two-tailed 0.0056 GW 3 retinasStudent t-test PD - Ctl 7 retinas F = 1.691, P = 0.5345 Unpairedtwo-tailed 0.1242 (n.s.) PD - GW 4 retinas Student t-test SP - Ctl 4retinas non Gaussian two-tailed Mann 0.0121 SP - GW 7 retinasdistribution Whitney test FIG. 12 a Ctl 15 retinas F = 5.546, P = 0.0047Unpaired two-tailed 0.0011 MCT 10 retinas Student t-test with Welch'scorrection b Ctl 6 retinas F = 11.79, P = 0.017 Unpaired two-tailed0.0231 MCT 6 retinas Student t-test with Welch's correction c Ctl 9retinas F = 1.807, P = 4504 Unpaired two-tailed 0.0035 TAK-875 8 retinasStudent t-test FIG. 13 a WT - Ctl 6 retinas F = 1.189, P = 0.8541Unpaired two tailed 0.0044 WT - GW 6 retinas Student t-test bVldlr^(−/−) - Ctl 6 retinas non Gaussian two-tailed Mann 0.0411Vldlr^(−/−) - GW 6 retinas distribution Whitney test c Ctl 3 experimentsF = 6.193, P = 0.2781 Unpaired two-tailed 0.0006 GW 3 experimentsStudent t-test d Complete media 3 experiments Two-way ANOVA Bonferonnipost-tests Complete vs No No glucose 3 experiments glucose: <0.01 e Ctl3 experiments F = 3.435, P = 0.4510 Unpaired two-tailed 0.0013 GW 3experiments Student t-test FIG. 14 a WT 13 retina non GaussianKruskal-Wallis with 0.0841 (n.s.) Vldlr^(−/−) 8 retinas distributionDunn's Multiple Vldlr^(−/−) + Dark 7 retinas Comparison TestVldlr^(−/−)/Ffar1^(−/−) 9 retinas b WT 7 retinas non GaussianKruskal-Wallis with 0.6177 (n.s.) Vldlr^(−/−) 8 retinas distributionDunn's Multiple Vldlr^(−/−) + Dark 9 retinas Comparison TestVldlr^(−/−)/Ffar1^(−/−) 8 retinas c, d, e 5 retinas/time pointDescriptive

Example 2: Correlation of the Number of RAP-Like Lesions and RetinalEnergy Demand

The number of RAP-like lesions was linked to photoreceptor energydemand. Rod photoreceptors consume 3-4 times more energy in darknessthan in light to maintain an electrochemical gradient required forphoton-induced depolarization known as the ‘dark current’(Okawa, H., etal., Curr Biol 18, 1917-1921 (2008)). Conversely, membrane turnover andvisual cycle activity are decreased in darkness (Okawa, H., et al., CurrBiol 18, 1917-1921 (2008)). Dark-raised Vldlr^(−/−) mice developed 1.5fold more vascular lesions than those raised in normal light/dark cycles(FIG. 1B), indicating that energy metabolism influenced neovasculardisease. Photoreceptors have been described to mature from the opticnerve outward towards the periphery. Energy consumption increases asphotoreceptors mature (P12-16), and at P16 the more mature centralretina possessed more RAP-like lesions (Wong-Riley, M. T. T. Eye Brain2, 99-116 (2010), and Trick, G. L. & Berkowitz, B. A. Prog Retin Eye Res24, 259-274 (2005)).

To determine whether Vldlr loss specifically in photoreceptors wassufficient to drive pathological vessels, remaining Vldlr wasselectively knocked down in photoreceptors (using AAV2-hRK) ofVldlr^(+/−) mice that possessed normal circulating fatty acid levels(Furukawa, et al., Cell 91, 531-541 (1997)) (FIGS. 5A-5I). Consistentwith energy deficiency, Vldlr^(−/−) photoreceptors exhibited swollenmitochondria (3D scanning electron microscopy and FIG. 1D; videos—notshown). Such results were further confirmed in videos (data not shown),which compared WT and Vldlr^(−/−) photoreceptor mitochondria, wherepseudo-colored mitochondria in WT or in Vldlr^(−/−) photoreceptors werevisualized by 3D reconstruction of scanning electron microscopy. InVldlr^(−/−) mice, a vascular lesion was also detected. Moreover,Vldlr^(−/−) retinas had lower ATP stores, (FIG. 1E), which confirmed anenergy deficit in the Vldlr^(−/−) mice.

Example 3: Fatty Acids Contributed to Retinal Energy Production inAddition to Glucose and were Likely Also Deficient in Vldlr^(−/−)Retinas

To explore the etiology of energy deficit fostering neovascularization,the contribution of FA and glucose to WT retinal energy production (FIG.6A) was examined. The long-chain FA palmitate fueled mitochondrialβ-oxidation in retinal explants, doubling oxygen consumption rates(OCR). Etomoxir-induced inhibition of FA transport into mitochondriaabrogated mitochondrial respiration, confirming that FA β-oxidationcontributes to retinal energy metabolism (FIGS. 2A-2B, and 6A-6C).

The contribution of glucose oxidation was further examined, and retinalglucose was also oxidized by mitochondria as efficiently as FA (FIGS.6D-6F). However, as reported by Warburg, Cohen and Winkler (Cohen, L. H.& Noell, W. K. J Neurochem 5, 253-276 (1960), and Winkler, B. S. J GenPhysiol 77, 667-692 (1981)), the vast majority of glucose (87%) wasconverted to lactate by glycolysis rather than oxidative phosphorylation(FIG. 6G). Unlike WT retinas, Vldlr^(−/−) retinas (with limited FAuptake) did not increase mitochondrial respiration when exposed topalmitate (FIG. 6H). FA, in addition to glucose, contributed to retinalenergy production, and may also be deficient in Vldlr^(−/−) retinas.

Quantification of Fatty Acid Uptake

In view of the possible role of Vldlr in retinal lipid energymetabolism, retinal FA uptake was quantified. Vldlr as reported washighly expressed in retinal photoreceptors (FIG. 7A) (Hu, W., et alInvest Ophthalmol Vis Sci 49, 407-415 (2008)). Retinal mid/long-chain FAuptake was reduced in Vldlr^(−/−) retinas. Serum turbidity reflectedhigher circulating lipid levels (FIGS. 7B and 7C). Triglycerides andmid/long-chain FA plasma levels (particularly palmitate) were elevatedin Vldlr^(−/−) mice (FIG. 6C, and FIGS. 7D and 7E). Importantly, theprocess of FA β-oxidation of lipids in mitochondria to produceacetyl-CoA was suppressed (FIG. 6D). In Vldlr^(−/−) retinas, totalacylcarnitine and free carnitine levels were reduced (FIG. 6E). Lowcytosolic FA levels were associated with decreased peroxisomeproliferative activated receptor (PPARα) expression (Lefebvre, P., etal., J Clin Invest 116, 571-580 (2006)), mainly in Vldlr^(−/−)photoreceptors (FIGS. 8A and 8B). PPARα is a key regulator of severalsteps of FA β-oxidation (Lefebvre, P., et al., J Clin Invest 116,571-580 (2006)); including Cpt1α that mediates internalization of FAinto mitochondria (FIG. 6B). Cpt1α was enriched in WT but suppressed inVldlr^(−/−) photoreceptors (FIG. 2F). A selective PPARα agonist(WY16463) used to enhance FA β-oxidation (Nakamura, M. T., et al., ProgLipid Res 53, 124-144 (2014)) reduced RAP-like lesions in Vldlr^(−/−)retina (FIG. 8C). Providing photoreceptor (661W) cells with palmitatealone or with PPARα agonist (GW9578) further increased mitochondrialrespiration by FA β-oxidation and not via uncoupling, since etomoxirabrogated the increase in respiration (FIGS. 9A-9D). Extracellularacidification rates, reflecting lactate production from glycolysis, werenot detectably affected by a PPARα agonist (FIGS. 9E and 9F). Furtherexploration of the role of PPARα in metabolic signaling in neovasculareye disease is warranted. Collectively, the findings indicate thatlipids are s an energy substrate retina, challenging the current dogmathat glucose is the only fuel of photoreceptors.

A compensatory upsurge in glucose uptake was expected to mitigate FAdeficiency in Vldlr^(−/−) retinas. Surprisingly, retinal glucose uptake(¹⁸F-FDG) was assessed by positron emitting tomography (PET) and retinal¹⁸F-FDG counts was reduced compared to WT (FIG. 2G and video dataobtained showing that FFA1 dictated glucose uptake in Vldlr^(−/−)retina, where ¹⁸F-FDG microPET/CT scan compared glucose uptakesimultaneously in WT, Vldlr^(−/−), Vldlr^(−/−)/Ffar1^(−/−) andFfar1^(−/−) mice (data not shown)), as was GLUT1 expression of the majorretinal glucose transporter Glut1 (FIGS. 2H and 2I), particularly inVldlr^(−/−) photoreceptors (FIG. 2H). In accord, carbohydrate metabolismwas the most significantly regulated pathway on a gene microarraycomparing Vldlr^(−/−) to WT retinas (FIG. 10A). The suppression ofpyruvate kinase (Pkm2), a critical enzyme of glycolysis was identifiedby the array and confirmed by qRT-PCR (FIG. 10B). Glut3 and 4 were notregulated (FIG. 10C). Hence, Vldlr^(−/−) retinas possessed both lipidand glucose uptake deficiencies (FIGS. 2A-2I), consistent with ageneralized energy shortage (FIGS. 1A-1E).

Example 4: Screening of Fatty Acid Sensing G-Protein Coupled MembraneReceptors

An abundance of lipids in Vldlr^(−/−) serum was postulated to signalthrough lipid sensors to reduce glucose uptake and help control fuelsupply to the retina (FIG. 3A). Known FA sensing G-protein coupledmembrane receptors (GPCR) in retina were screened. FFA1 was the mostabundantly expressed FA receptor in WT and increased further inVldlr^(−/−) retinas, particularly in photoreceptors (FIGS. 38 and 3C).FFA1, first discovered in the pancreas (Itoh, Y., et al. Nature 422,173-176 (2003)), governs glucose transport and insulin secretion(β-islet cells) (Kebede, M., et al. Diabetes 57, 2432-2437 (2008), andAlquier, T., et al. Diabetes 58, 2607-2615 (2009)). High pancreatic FFA1expression suppressed expression of the main endocrine pancreas glucosetransporter, Glut2 (Steneberg, P., et al., Cell Metabolism 1, 245-258(2005)). Ffar1 has also been localized in brain, where its function isnot well defined Honord, J.-C., et al. Arterioscler Thromb Vasc Biol 33,954-961 (2013)), and Briscoe, C. P., et al. The J Biol Chem 278,1130311311 (2003)). In WT and Vldlr^(−/−) retinas a FFA1 agonist(GW9508) was identified that suppressed the expression of Glut1 (Gospe,S. M., et al. J Cell Sci 123, 3639-3644 (2010)) and retinal glucoseuptake (FIG. 3D and FIG. 11A). Importantly, treatment with FFA1 agonist(GW9508) more than doubled the number of RAP-like lesions in Vldlr^(−/−)retinas compared to controls (FIG. 3F). FFA1 binds lipids comprisingmore than 6 carbons Briscoe, C. P., et al. The J Biol Chem 278,1130311311 (2003)). Vldlr^(−/−) mice treated with FFA1 lipid agonistsmedium chain triglycerides (MCT; 8-10 carbons) (Briscoe, C. P., et al.The Journal of biological chemistry 278, 1130311311 (2003)) or a secondFFA1 selective agonist TAK-875 (Naik, H., et al. J Clin Pharmacol 52,1007-1016 (2012)) increased Glut1 suppression and more RAP-like lesionsversus controls (FIGS. 12A-12C). Deletion of Ffar1 in Vldlr^(−/−) miceraised retinal glucose uptake (FIG. 3G and video data as described above(data not shown)) and Glut1 expression towards WT and Ffar1^(−/−) levels(FIG. 3H, and FIGS. 11B-11C), with fewer RAP-like lesions inVldlr^(−/−)/Ffar1^(−/−) mice (FIG. 3I). In vitro knock down of Ffar1 ortreating cells with MEK/ERK inhibitor (PD98059) prevented Glut1suppression by GW9508 in photoreceptors (661W; FIGS. 11D-11F).Therefore, Ffar1 may act as nutrient sensor, coupling mitochondrialmetabolism with circulating substrate availability.

Example 5: Lipid/Glucose Fuel Insufficiency in Retina, can DriveAberrant Angiogenesis in the Normally Avascular Photoreceptor Layer

It was hypothesized that photoreceptors challenged by a dual glucose andlipid fuel substrate deficiency would signal to increase vascularsupply, in an attempt to restore energy homeostasis (FIG. 4A). Hypoxiahas been assumed to be the main driver of angiogenesis, but inadequatenutrient availability to tissue might also control blood vessel growth.A reduction in pyruvate levels, metabolic intermediates feeding into theKrebs (TCA) cycle from decreased glucose uptake and glycolysis (FIG. 4B)and also less acetylcarnitine from decreased FA uptake and β-oxidation(FIG. 4C) were identified as associated with reduced production ofessential intermediates, such as α-ketoglutarate (α-KG was lower, FIG.4D).

Together with oxygen, α-KG is a necessary co-activator ofpropyl-hydroxylase dehydrogenase (PHD) that tags HIF1α for degradationby proline hydroxylation (Kaelin, W. G. Cold Spring Harb Symp Quant Biol76, 335-345 (2011)). Less hydroxyproline was detected in Vldlr^(−/−)retinas, consistent with reduced PHD activity (FIG. 4E). Indeed,reduction in retinal glucose uptake by FFA1 agonist GW9508 and glucosestarvation in photoreceptors (661W) was associated with Hif1αstabilization (FIGS. 13A-13C) and Vegfa secretion (FIGS. 13D and 3E). Invivo, Hif1α stabilization (FIG. 4F) in Vldlr^(−/−) photoreceptors (FIG.4G) was associated with Vegfa production (FIGS. 4H and 4I). Deletion ofFfar1 in Vldlr^(−/−) retinas suppressed HIFα stabilization and Vegfasecretion (FIGS. 4F and 4H), thereby leading to fewer vascular lesions(FIG. 3I). Consistent with the previous results Hua, J., et al.Investigative ophthalmology & visual science 52, 2809-2816 (2011)), miceengineered to secrete Vegfa in photoreceptors developed retinalangiomatous proliferation comparable to Vldlr^(−/−) mice (Ohno-Matsui,K., et al. The American journal of pathology 160, 711-719 (2002)); Vegfafrom photoreceptors was therefore sufficient to promote RAP-likelesions. Importantly, oxidative stress associated with an energy crisislikely also directly stabilized Hif1α and promoted Vegfa secretion inVldlr^(−/−) photoreceptors (Dorrell, M. I., et al. J Clin Invest 119,611-623 (2009), Chen, Y., et al. Microvasc Res 78, 119-127 (2009), andZhou, X., et al., PloS One 6, e16733 (2011)), potentially contributingto vascular lesions. However, macrophages, often implicated in theetiology of AMD, were not associated with the onset of development ofnascent RAP-like lesions, surrounding only mature vascular lesions(FIGS. 14A-14D). Translating these findings to human disease, highervitreous VEGF levels were detected in AMD/RAP human subjects compared tocontrols (macular hole; FIG. 4J). These findings indicated thatlipid/glucose fuel insufficiency in retina, in part through reduction ofKrebs cycle metabolite α-KG, could drive aberrant angiogenesis in thenormally avascular photoreceptor layer.

In the retina, the ability to use both lipids and glucose as fuel mightbe beneficial during periods of high fuel need or fuel deprivation.Fasting liberates FA from adipose tissue that is used by high-energyconsuming organs capable of FA β-oxidation, such as heart, and perhapsretina (FIGS. 2A-2F, FIGS. 6A-6C, and FIGS. 9A-9D). Indeed, FAβ-oxidation disorders were previously identified as associated withretinopathy (Fletcher, et al., Molecular Genetics and Metabolism 106,18-24 (2012)). Tissues that use lipid as fuel curb glucose uptake duringstarvation (Mantych, G. J et al., Endocrinology 133, 600-607 (1993), andFerrannini, E., et al. J Clin Invest 72, 1737-1747 (1983)). The capacityto sense nutrient availability and adapt fuel uptake could improvemetabolic efficiency.

G-protein coupled receptors (GPCR) are known membrane sensors of aminoacids, glucose and lipids (Wauson, E. M. et al. Mot Endocrinol 27,1188-1197 (2013)). Here, FFA1 was shown to be a metabolic sensor of FAavailability, which controls glucose entry into the retina (FIGS.3A-3I). Long-term suppression of glucose entry by FFA1 in photoreceptors(perhaps secondary to circulating lipids) likely contributed toage-related mitochondrial dysfunction in AMD or MacTel. Retinal effectsof FFA1 agonists considered for the treatment of type II diabetes shouldbe carefully monitored, particularly in older individuals at increasedrisk of AMD.

SUMMARY

Mitochondrial metabolism may contribute to pathological angiogenesis inother diseases, such as cancer. To provide building blocks forproliferation, tumors promote angiogenesis at the cost of efficient ATPproduction by the Warburg effect (Warburg, O. Science 123, 309-314(1956)). In suppressing mitochondrial oxidative phosphorylation, tumorcells may generate less α-KG (Zhao, S., el al. Science 324, 261-265(2009)) (or accumulate competing metabolites) (Kaelin, W. G. Cold SpringHarb Symp Quant Biol 76, 335-345 (2011)). This inhibitsprolyl-hydroxylase dehydrogenase (PHD) with ensuing HIF1α stabilization,driving tumor angiogenesis required for growth. The findings indicatethe importance of mitochondrial fuel starvation as a driver ofangiogenesis, matching energy demands with vascular supply. With adecline in mitochondrial function with age, this process may contributeto pathological angiogenesis in diseases of aging retina.

In summary, lipid uptake and lipid β-oxidation are curtailed inVldlr^(−/−) retinas. Increased circulating FA can activate FFA1,associated with decreased retinal glucose uptake and decreased Krebcycle intermediate α-KG. Hifα is stabilized and Vegfa secreted byVldlr^(−/−) photoreceptors, giving rise to pathologic RAP-likeneovessels. This study uncovered three important novel mechanismscontributing to retinal physiology and neovascular AMD/RAP:

-   -   (1) lipid β-oxidation is an energy source for the retina,    -   (2) FFA1 is an important nutrient sensor of circulating lipids        that controls retinal glucose entry to match mitochondrial        metabolism with available fuel substrates, and    -   (3) nutrient scarcity is a driver of retinal pathological        angiogenesis.        These pathways may be important for discovery of new        therapeutics.

Example 6: Use of FFA1 Inhibitors to Treat Neural Cell (e.g., RetinalCell) Diseases or Disorders

In this example, a subject having or at risk of developing a neural cell(e.g., retinal cell) disease or disorder characterized by angiogenesis(e.g., a subject having or at risk of RAP vascular lesions, e.g., asubject having or at risk of MacTel and/or AMD) is administered apharmaceutical composition comprising a FFA1 inhibitor. Preventionand/or improvement of the neural cell (e.g., retinal cell) disease ordisorder characterized by angiogenesis is monitored both before andafter administration of the FFA1 inhibitor to the subject. The FFA1inhibitor (e.g., RNAi and/or small molecule) is administeredsystemically or locally to the subject (e.g., via intraocular injectionto a subject). Prophylactic and/or therapeutic efficacy of the FFA1inhibitor in the subject is thereby identified.

Example 7: Identification of Novel FFA1 Inhibitors

In this example, photoreceptor cells (e.g., 661W cells) are grown invitro, optionally in an array format. Cells (optionally having amutation or deletion of the very low-density lipoprotein receptor(Vldlr) gene) are contacted with libraries of test compounds, andcellular glucose uptake is monitored. Test compounds that reproduciblyaffect a significant increase in glucose uptake are thereby identifiedas candidate novel inhibitors of FFA.

Contemplated assays for identification of new FFA1 inhibitors includeradioactive calorimetric assays, as well as non-radioactive calorimetricassays to measure glucose uptake. The assays are optionally optimizedfor high throughput analysis. For example, a non-radioactive assayincludes a colorimetric glucose uptake assay kit (e.g., Abcam productnumber ab136955). Additionally, a glucose uptake cell-based assay kit(Cayman chemical Item No 600470) is used to measure glucose uptake usinga fluorescent deoxyglucose analog. In yet another example, the FFA1inhibitors are identified through the cell surface representation of theglucose transporter, Glut1 (e.g., ELISA-based assays and/or geneexpression assays).

Example 8: Use of FFA1 Inhibitors to Treat Neurodegeneration

In this example a subject having or at risk of developingneurodegeneration (characterized by a loss in function of neurons andalso including death of neurons) is administered a pharmaceuticalcomposition comprising a FFA1 inhibitor. Prevention and/or improvementof the neurodegeneration is monitored both before and afteradministration of the FFA1 inhibitor to the subject. Prophylactic and/ortherapeutic efficacy of the FFA1 inhibitor in the subject is therebyidentified.

Example 9: Use of FFA1 Inhibitors to Treat Cancer

In this example, a subject having or at risk of developing cancer isadministered a pharmaceutical composition comprising a FFA1 inhibitor.Prevention and/or improvement of the cancer is monitored both before andafter administration of the FFA1 inhibitor to the subject. Prophylacticand/or therapeutic efficacy of the FFA1 inhibitor in the subject isthereby identified. In some examples, the inhibitors alter themetabolism of malignant cells, thereby treating the subject.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for treating or preventing angiogenesis in neural cells of asubject, treating or preventing cancer in a subject, and/or treating orpreventing retinal angiomatous proliferation (RAP) vascular lesions in asubject, the method comprising: (a) identifying a subject having or atrisk of neural cell angiogenesis, and/or having or at risk of developingcancer, and/or having or at risk of developing RAP vascular lesions; and(b) administering a FFA1 inhibitor to the subject, thereby treating orpreventing angiogenesis in the neural cells of the subject, treating orpreventing cancer in the subject, and/or treating or preventing RAPvascular lesions in the subject.
 2. The method of claim 1, wherein theneural cells are retinal cells, optionally photoreceptor cells. 3.(canceled)
 4. The method of claim 1, wherein the subject has maculartelangiectasia (MacTel) or neovascular age-related macular degeneration(AMD).
 5. The method of claim 1, wherein the cells of the subject areimpaired for lipid uptake, as compared to the cells of an appropriatecontrol subject.
 6. The method of claim 1, wherein the subject hasdyslipidemia or mitochondrial dysfunction.
 7. The method of claim 1,wherein the FFA1 inhibitor is a small molecule antagonist or an RNAiagent.
 8. The method of claim 1, wherein the FFA1 antagonist is GW1100.9. The method of claim 1, wherein the FFA1 inhibitor is administered tothe eye of the subject.
 10. The method of claim 9, wherein the FFA1inhibitor is administered by intravitreal injection.
 11. The method ofany claim 1, wherein administering the FFA1 inhibitor enhances GLUT1expression in the retinal cells of the subject.
 12. A method forincreasing glucose uptake in a retinal cell, the method comprisingobtaining a retinal cell and contacting the retinal cell with a FFA1inhibitor, thereby increasing glucose uptake in the retinal cell. 13.The method of claim 12, wherein the retinal cell is a retinal cell invitro.
 14. The method of claim 12, wherein GLUT1 expression is enhancedin the retinal cell contacted with the FFA1 inhibitor.
 15. A method foridentifying a test compound as a FFA1 inhibitor, the method comprisingcontacting a retinal cell with a test compound; and measuring glucoseuptake in the retinal cell, wherein measurement of increased glucoseuptake in the retinal cell in the presence of the test compoundidentifies the test compound as a FFA1 inhibitor.
 16. The method ofclaim 15, wherein the retinal cell has a mutation or deletion of thevery low-density lipoprotein receptor (Vldlr) gene that suppresses fattyacid uptake in the retinal cell.
 17. The method of claim 15, whereinGLUT1 expression is enhanced in the retinal cell contacted with the testcompound.
 18. A method for treating or preventing a vascular disease ofthe eye, retinal degeneration and/or cancer in a subject, the methodcomprising: (a) identifying a subject having or at risk of developing avascular disease of the eye, retinal degeneration and/or cancer; and (b)administering a GPR84 inhibitor, or a GPR120 inhibitor to the subject,thereby treating or preventing the vascular disease of the eye, retinaldegeneration and/or cancer in the subject.
 19. The method of claim 18,wherein the GPR84, or the GPR120 inhibitor is a small moleculeantagonist or an RNAi agent.
 20. The method of claim 18, wherein theGPR84 inhibitor is GLPG1205, or the GPR120 inhibitor is4-Methyl-N-9H-xanthen-9-yl-benzenesulfonamide.
 21. The method of claim18, wherein the vascular disease of the eye is age-related maculardegeneration (AMD) or retinopathy of prematurity (ROP). 22.-25.(canceled)